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Use of Aloe vera–based extender for chilling and freezing collared peccary (Pecari tajacu) semen
Accepted Manuscript Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari tajacu) semen A.L.P. Souza, G.L. Lima, G.C.X. Peixoto, A.M. Silva, M.F. Oliveira, A.R. Silva PII:
S0093-691X(16)00008-X
DOI:
10.1016/j.theriogenology.2016.01.007
Reference:
THE 13475
To appear in:
Theriogenology
Received Date: 14 January 2015 Revised Date:
4 January 2016
Accepted Date: 4 January 2016
Please cite this article as: Souza A, Lima G, Peixoto G, Silva A, Oliveira M, Silva A, Use of Aloe Verabased extender for chilling and freezing collared peccary (Pecari tajacu) semen, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.01.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari
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tajacu) semen
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ALP Souzaa, GL Limaa, GCX Peixotoa, AM Silvaa, MF Oliveiraa, AR Silvaa*
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a
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do Semi-Árido (UFERSA), Mossoró, RN, Brazil
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*Corresponding author. Tel.: 55 84 33178374. E-mail address: [email protected]
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(AR Silva).
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9 Abstract
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Laboratory of Animal Germplasm Conservation - LCGA, Universidade Federal Rural
As an alternative for the conservation of collared peccary semen, this research
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aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen
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chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in
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Tris plus egg yolk—EY (20%) or AV extract (20%)—and chilled at 5 ºC. In both
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treatments, an adequate semen conservation was achieved and values closer to 40%
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motile sperm with viability and osmotic response ranging from 20 to 40%, and normal
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morphology of 80% were found after 36 h of storage. Moreover, 12 other ejaculates
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were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%).
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Samples were frozen in liquid nitrogen and thawed after one week. After thawing, all
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the treatments containing EY or AV provided similar values for sperm morphology,
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viability, osmotic response, membrane integrity, sperm motility, ALH, BCF, and rapid,
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low, and static subpopulations, but the highest values for STR and the lowest values for
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VCL were found using 20% AV (P < 0.05). In conclusion, we demonstrate that Aloe
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vera extract at a 20% concentration could be used as an alternative substitute to egg
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yolk in the formulation of Tris extenders for collared peccaries’ semen chilling or
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freezing.
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Keywords: Tayassu tajacu; sperm chilling; sperm freezing; Aloe vera; cryoprotectant.
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29 1. Introduction
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With the advent of semen conservation, the possibility of germplasm storage
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enables posterior manipulation and use of assisted techniques such as in vitro
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fertilization or artificial insemination [1]. Despite this, the decrease in temperature rates
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during semen chilling and freezing procedures is critical to maintain the sperm quality
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after rewarming, with the sperm sensitivity varying depending on the species [2].
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To reduce the cryoinjuries, several substances have been used as cryoprotectants,
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and the hen’s egg yolk has showed satisfactory results for both domestic [3, 4, 5] and
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wild species [6, 7, 8]. Egg yolk presents beneficial effects on sperm cryopreservation as
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a protectant of the plasma membrane and acrosome against temperature-related injury
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[3]. It is believed that the phospholipids, cholesterol, and low-density lipoproteins
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present in egg yolk may be factors that provide protection to the sperm against cold
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shock during the freeze–thaw process [4]. However, because it is a product of animal
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origin, there is the possibility of bacterial contamination [9], which contributes to the
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difficulty of exchanging semen among different regions or countries [5]. Furthermore,
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egg yolk can interfere with microscopic observations or biochemical assays, as it
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contains granular material of the same size and shape as the sperm [10].
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Several authors search for an alternative medium that is free of animal products
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for semen conservation. In this sense, the Aloe vera appears as an alternative from
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vegetal origin for this purpose, as it constitutes some substances that can act as the
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conventional cryoprotectants [11]. Along the years, Aloe vera has been used in the
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cosmetic and toiletry industry due to its curative and therapeutic properties [12]. In
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addition, several studies show the positive action of Aloe vera extract on
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spermatogenesis [13]. The use of Aloe vera extract for an effective semen conservation,
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however, is restricted to the caprine semen chilling [14] and ovine semen freezing and
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artificial insemination [15]. Therefore, it presents a potential as a good alternative for
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sperm cryoprotection and should be better explored and extrapolated to other species,
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including wild animals.
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The collared peccary (Pecari tajacu) is a wild species whose meat and pelt has
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great international demand, but the number of individuals in some of its natural habitats,
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such as the Caatinga and Atlantic Forest, is critically decreasing due to poaching, which
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has necessitated them to be bred under captivity [16]. The development of assisted
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techniques that make the conservation of collared peccary viable besides the
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implementation of germplasm banks is very important for their multiplication, which
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has generated some studies on its semen conservation. It was demonstrated that their
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semen could be chilled at 17 ºC for only 24 h by using the Beltsville Thawing Solution
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as extender [17]. At freezing, however, several studies demonstrated that the sperm
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quality could be efficiently preserved using Tris [18] or coconut water extenders [6] and
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both plus egg yolk. In order to present an alternative cryoprotectant for the conservation
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of its semen, this research aims at evaluating the use of Aloe vera extract as a
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cryoprotectant for the peccaries’ semen chilling and freezing.
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2. Materials and methods
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The ethics committee of the Universidade Federal Rural do Semi-Árido—
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UFERSA has approved the experimental protocols and the animal care procedures
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adopted (process no. 23091.0253/114). The reagents used in this study were obtained
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from Sigma-Aldrich (St. Louis, MO, United States), except when cited.
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2.1. Animals and semen collection
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Samples were obtained from 12 sexually mature male collared peccaries with an
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average (SD) age and weight of 40.7 ± 1.6 mo and 22.5 ± 2.8 kg, respectively. Five of
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the individuals served as semen donors for the semen chilling and freezing, being
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subjected to two electroejaculation sessions on a 30-day interval. The other seven
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animals were used as semen donors for only the semen freezing.
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The animals belonged to the Center of Multiplication of Wild Animals,
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UFERSA, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W).
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The climate is typically semiarid, with an average annual temperature of 27 °C. The
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animals were isolated from the females at 6 months before the commencement of the
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study and maintained under a 12-h natural photoperiod. They were maintained outdoors
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in groups of six in paddocks (20 m to 3 m) with a covered area of (3 to 3 m) and fed
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sow food and fruits (with water available ad libitum).
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Animals were fasted 12 h before the experiments began. They were then
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physically restrained using a hand net and anesthetized using intravenous administration
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of propofol (Propovan®, Cristalia, Fortaleza, Brazil), which was given as a bolus (5
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mg/kg) [19]. They were kept in lateral recumbency, and the semen was collected with
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an electroejaculator (Autojac®, Neovet, Campinas, SP, Brazil) that was connected to a
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12 V source. The stimulatory cycle comprised 10 stimuli in each voltage, starting from
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5 V, followed by a voltage increase in steps from 1 V to 12 V. Each electrical stimulus
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lasted 3 s, with intermittent breaks of 2 s. The stimuli cycle was maintained for a 10 min
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duration from the beginning of the procedure. The electroejaculator probe was 15 x 1.3
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cm, and it was inserted 12 cm into the rectum. Semen was collected into plastic tubes
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and immediately evaluated [18].
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2.2. Experimental design
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The execution of the study was divided into two steps. At first, we verified the
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effect of Aloe vera (AV) extract on the short-term preservation at 5 ºC of collared
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peccaries’ semen by comparing it with the egg yolk (EY), both at a 20% concentration
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added to the Tris extender. Afterward, we verified the effect of AV extract at different
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concentrations (5, 10, and 20%) added to the Tris extender for the collared peccaries’
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semen freezing. In both experiments, we used the Tris-based extender formulation
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previously described for collared peccaries [18].
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2.3. Extraction of Aloe Vera
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The plant Aloe vera was cultured in a sandy clay soil type at the UFERSA
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campus, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W),
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under a typical semiarid climate, with an average annual temperature of 27 °C. In order
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to obtain the crude extract of Aloe vera, we extracted the parenchyma of its leaves as a
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colorless gel. It was filtered through a sieve; then, it was packed in a glass container till
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its use.
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2.4. Semen chilling
126 Semen samples that were collected after electroejaculation of 5 individuals were
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divided into two aliquots. The first was diluted in Tris plus EY (20%), and the other was
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diluted in Tris plus AV extract (20%), reaching a concentration of 100 x 106 sperm/mL.
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Samples were evaluated immediately after dilution (0 h) for sperm total motility,
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viability, osmotic response, and morphology. Then, samples were stored in the water
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jacket at 27°C and equilibrated for 40 min to reach 15°C in a biological incubator
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(Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to establish a
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temperature at 5 ºC for 30 min. Samples were rewarmed at 37 ºC and reevaluated every
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12 h till 36 h.
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2.5.Semen freezing
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Semen was frozen according to a methodology previously developed for
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peccaries [4]. Samples derived from 12 individuals were divided into 4 aliquots. These
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were diluted in Tris extender supplemented with EY (20%), as a control group, or AV
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extract at 5, 10, or 20%. All the dilutions were conducted at room temperature (27 °C),
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and the samples were then equilibrated for 40 min to reach 15°C in a biological
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incubator (Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to
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establish a temperature at 5 ºC for 30 min. At that point, the sample was added to the
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extender with 6% glycerol (also at 5ºC), which resulted in a final concentration of 3%
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glycerol in the extender and 100 x 106 sperm /mL. Finally, the samples were packed in
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0.25-mL plastic straws (IMV Technologies; L’Aigle, France) that were placed
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horizontally in an insulated box for 5 min, at 5 cm above the nitrogen (N2) vapors, and
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then plunged into liquid N2 for storage at −196 ºC, following a fast freezing rate at −40
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ºC/min. After 1 week, frozen straws were thawed by immersion in a water bath at 37°C
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for 1 min [6]. Samples were evaluated for sperm motility parameters, viability,
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morphology, osmotic response, and membrane integrity.
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154 2.6. Semen evaluation
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Fresh ejaculates were evaluated for aspect, color, and volume. The sperm
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concentration was determined using a Neubauer counting chamber [20]. After
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electroejaculation, rewarming, and freezing–thawing, samples were evaluated for sperm
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morphology, viability, and osmotic response. Bengal Rose–stained smears were
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prepared to evaluate sperm morphology using light microscopy (1000×), counting 200
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cells per slide [21]. Sperm viability was determined by analyzing a slide stained with
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bromophenol blue under light microscopy (400×), counting 200 cells per slide [22].
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Functional integrity of the sperm membrane was verified by evaluating the sperm
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osmotic response under a hypo-osmotic swelling (HOS) test, using distilled water (0
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mOsm/L) as the hypo-osmotic solution. A total of 200 sperms were examined, and
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those with a swollen coiled tail were considered as presenting a functional membrane.
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[23]. For the evaluation of the plasma membrane integrity in frozen–thawed samples,
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we used a fluorescent solution that contained fluorophores diacetate 6-carboxy-
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fluorescein (C-FDA) and propidium iodide (PI). Samples were incubated for 10 min and
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evaluated by epifluorescence microscopy (Episcopic Fluorescent attachment “EFA”
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Halogen Lamp Set, Leica, Kista, Sweden). For each sample stained with CFDA/PI, 200
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sperms were counted and classified as having or not having an intact membrane (based
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on color) [24].
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2.7. Computer-assisted sperm assessment
177 Sperm motility parameters were analyzed by computer-assisted sperm
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assessment (IVOS 7.4G, Hamilton-Thorne ResearchTM, Beverly, MA, USA) in fresh,
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rewarmed, and frozen–thawed semen. The settings of the instrument were validated for
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collared peccaries semen before analysis, including temperature 37 ºC; 60 frames/s;
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minimum contrast, 45; straightness threshold, 30%; low-velocity average pathway
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(VAP) cutoff, 10 µ/s; and medium VAP cutoff, 30 µ/s. Five independent and
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nonconsecutive microscopic fields were randomly selected and scanned. The following
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endpoints were analyzed: number of counted cells, total motility (%), VAP (µm/s),
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velocity straight line (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of
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lateral head (ALH; µm), beat cross frequency (BCF; Hz), straightness (STR;%), and
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linearity (LIN;%). According to low VAP cutoff (LVV) and medium VAP cutoff
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(MVV), the overall sperm population was subdivided into four categories: rapid, with
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VAP > MVV; medium, with (LVV < VAP
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the proportion of cells that were not moving [25]. For a trustable evaluation of the
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sperm tracks, the Edit Tracks Option of the IVOS 7.4G system was used to exclude the
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debris derived from the extenders. A further dilution in salt solution (1:1) was
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conducted only if necessary.
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2.8. Statistical analysis
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Results were expressed as mean ± SEM. All analyses were performed using the
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Statview 5.0 software (SAS Institute Inc., Cary, NC, USA). The data were first
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examined for normality by the Shapiro–Wilk test and for homoscedasticity by the
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Levene’s test. In view of some data that did not present a normal distribution, an arcsine
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transformation was conducted for percentage values. The effect of incubation time (0,
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12, 24, and 36 h) during chilling on sperm parameters was evaluated by variance
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analysis (ANOVA) for repeated measures. Comparisons among treatments for chilled or
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frozen–thawed semen were evaluated by ANOVA, followed by the Fisher PLSD test.
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Statistical significance was set at P < 0.05.
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3. Results
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Fresh ejaculates presented a watery aspect, white color, and a volume of 2 ± 0.4
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mL with a concentration of 259.2 ± 29.3 x106 sperm/mL. Average values of 97.5 ±
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1.0% motile sperm, being 83.4± 23% viable and 88.1± 1.5% morphologically normal,
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with 84.5 ± 3.8% osmotic response were found in fresh samples.
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3.1. Semen chilling
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On evaluating the peccary chilled semen during 36 h, no statistical differences
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were found between samples diluted in Tris plus EY or AV extract for sperm viability,
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morphology, and osmotic response (Figure 1) at any time. However, we verify a
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significant linear reduction in the preservation of sperm viability and osmotic response
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during the experiment for both treatments (P < 0.05) that presented values between 20
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and 40% at 36 h.
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For CASA evaluation, no samples were rediluted. The kinematic parameters of
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sperm motility generated by CASA (Table 1) confirmed the similarity between the
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cryoprotectants used for cooling the semen of peccary for 36 hours; however, a
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significant decrease on the values for total motility was observed in the use of AV after
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36 h (P < 0.05).
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After thawing (Table 2), all the treatments containing EY or AV provided
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similar values for sperm morphology, viability, osmotic response, and membrane
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integrity.
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Regarding CASA evaluation, the samples diluted in Tris-EY presented large
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amounts of debris, demanding a long time for its exclusion using the tracks edition
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option; however, the redilution was only necessary in 3 of the 12 frozen–thawed
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samples. In contrast, a very low debris formation was observed when using the AV
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extract in any concentration, where a tracks edition was rarely needed and a redilution
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unnecessary.
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CASA reveals (Table 3) that all the treatments provided a similar preservation of
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sperm motility, ALH, BCF, and rapid, low, and static subpopulations, but the highest
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values for STR and the lowest values for VCL were found using 20% AV (P < 0.05).
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Some kinetic parameters as STR and LIN were significantly reduced in the groups
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containing 5 and 10% AV compared to those containing 20% AV (P < 0.05). Moreover,
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the group containing EG presented values for medium subpopulation lower than those
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found for 20% AV (P < 0.05).
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4. Discussion
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As previously shown for caprine [14], the AV extract showed an effective
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cryoprotective effect on peccaries’ semen chilling, providing results similar as the
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extender containing EY. Chauhan et al. [26] revealed the Aloe vera’s chemistry and
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reported the presence of more than 200 different biologically active substances,
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including vitamins, minerals, enzymes, sugars, anthraquinones or phenolic compounds,
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lignin, saponins, sterols, amino acids, and salicylic acid. In such components, the
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presence of polysaccharides such as pectin, hemicellulose, glucomannan, and
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acemannan is highlighted, with the majority being glucose and mannose derivatives
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[27]. These sugars are known as important energy sources for the sperm [28], and they
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could have contributed to the peccary sperm’s short-term preservation. Moreover, the
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presence of substances presenting antioxidant capability such as polyphenols, indoles,
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and alkaloids was recently detected in the AV extract [29]. This property could add to
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the prevention of the sperm oxidative damage derived from the hypothermic storage of
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peccaries’ liquid semen.
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In this study, we demonstrate that the peccary semen could be efficiently stored
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at 5 ºC during 36 h in the use of a Tris extender supplemented with EY or AV. These
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results are better than those previously reported for the same species, in which values of
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only 22% motile sperm were verified after 24 h storage at 17 ºC using the Beltsville
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Thawing Solution (BTS) [17]. It is known that the use of a storing temperature varying
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from 15 to 20°C maintains the cellular activity, making sperm more sensitive to toxic
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metabolic agents [30]. Therefore, the storage of semen in temperatures closer to 4°C, as
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used in the present study, promotes a reduction in the sperm metabolism, saving its
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energy reserves, thus contributing to a good preservation of sperm lifespan [30].
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Regarding peccaries’ semen freezing, we verify that extenders containing AV
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extract provide values for post thawing sperm parameters similar to those previously
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reported for the same species in other studies that used EY plus Tris [18] or coconut
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water [6] extenders. The use of 20% AV, however, provided the best preservation of
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STR after thawing. Recently, this parameter, a marker for sperm progression used for
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describing sperm swimming patterns [31], was positively related to the capability of
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peccary spermatozoa to interact with swine oocytes [32]. In contrast, the same group,
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20% AV, provided the lowest values for VCL, which is the motion parameter
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considered as the main determinant of hyperactivated motility [33]. Therefore, we can
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assume that the use of 20% AV would be more effective than 20% EY in preventing
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sperm hyperactivation after freezing–thawing procedures.
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The reduction of AV concentration from 20% to 10 or 5% was not effective in
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preserving STR or LIN values, and we could not discard the existence of any
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detrimental activity on the sperm when using AV concentrations higher than 20%. This
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statement is based on the fact that Gutiérrez et al. [15] tested the AV extract
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concentrations, varying from 10 to 70%, which were added to coconut water extender
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for ovine semen freezing. They demonstrate that the best semen preservation is
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achieved when this extract is used from a 20 to 50% concentration, but if the
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concentration is increased above 50%, it will present significant detrimental effects on
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semen quality.
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cryoprotective effect remains unknown. It is known that it contains folic acid and zinc
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[34] that act as antioxidants, thus improving the semen quality by reducing semen
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apoptosis [35]. Furthermore, it contains various polysaccharides [27], which, besides
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serving as an energy source, are known to present some extracellular cryoprotective
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effects, thus contributing to the sperm membrane stability [36]. In addition, AV extract
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also contains phenol, saponin, and anthraquinones components that have antiviral and
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antifungal properties [26], and it was recently demonstrated to have antibacterial
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property against gram-positive and gram-negative pathogens [29]. In this sense, the AV
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extract could be used as an alternative to EY because EY might contribute to possible
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risks of contamination, which in turn results in the difficulty of exchanging semen
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among different regions or countries [10]. Moreover, the amount of debris formation
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provoked by AV use does not impair the peccary semen evaluation. This statement
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would be an enormous advantage for the procedure, thus facilitating the manual debris
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exclusion by using the CASA option for selecting and zooming individual tracks [37].
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Moreover, AV use would avoid or reduce the need for additional post thawing dilution
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in isosmotic substances that reduce the debris concentration, thus facilitating the
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computer analyses [38, 39].
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In conclusion, we demonstrate that the Aloe vera extract could be efficiently used
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as a substitute for egg yolk in the formulation of Tris extenders for collared peccaries’
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semen chilling or freezing. Furthermore, the chilling process at 5 ºC provided adequate
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sperm motility till 36 h by using egg yolk or Aloe vera (20%) in Tris extender. Finally,
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we suggest the use of Aloe vera extract at 20% concentration in Tris extender for
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collared peccaries’ semen freezing.
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Acknowledgements
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The authors thank the Brazilian Council of Scientific Development - CNPq (Process n°
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552327/2011-5) for financial support; and CEMAS/UFERSA for providing the animals.
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Ana Liza Paz Souza, Gislayne Christianne Xavier Peixoto, and Andreia Maria Silva
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were recipients of a grant from CAPES. Moacir Franco de Oliveira and Alexandre
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Rodrigues Silva were recipients of a grant from CNPq.
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2004;32:887–893.
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[31] Peña A, Linde-Forsberg C. Effects of Equex, one or two step dilution and two
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freezing thawing rates on post-thaw survival of dog spermatozoa. Theriogenology
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2000;54:859–75.
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[32] Campos LB, Souza ALP, Pereira AF, Silva AR. Estimating the fertilizing ability of
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collared peccaries (Pecari tajacu) sperm by analyzing its interactions with swine
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oocytes. Anim Reprod 2015;12:551.
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[33] De-Geyter C, De-Geyter M, Koopers B, Nieschiaq E. Diagnostic accuracy of
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computer-assisted sperm motion analysis. Hum Reprod 1998;13:2512–2520.
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[34] Shahraki A, Mojahed AS, Afshar-Goli J. The effects of hydroalcoholic extract of
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Aloe vera gel on spermatogenesis of adult male rats. Int J Biosci 2014;5:158–165.
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[35] Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm
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GW, Behne D. Identification of a specific sperm nuclei selenoenzyme necessary for
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Polysaccharide on boar spermatozoa quality following freezing-thawing. Cryobiology
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2009;59:244–249.
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[37] Mortimer ST, van der Horst G, Mortimer D. The future of computer-aided sperm
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analysis. Asian J Androl 2015;15:545–553.
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[38] Anel L, Gomes-Alves S, Alvarez M, Borragan S, Anel E, Nicolas M, Martinez-
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Pastor F, Paz P. Effect of basic factors of extender composition on post-thawing quality
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of brown bear electroejaculated spermatozoa. Theriogenology 2010;74:643–651.
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[39] Pillet E, Duchamp G, Batellier F, Beaumal V, Anton M, Desherces S, Schimidt E,
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Magistrini M. Egg yolk plasma can replace egg yolk in stallion freezing extenders.
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Theriogenology 2011;75:105–114.
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Values (means ± SEM) for kinematic parameters of sperm motility in collared
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Table 1:
peccaries (Pecari tajacu) chilled semen diluted in Tris plus egg yolk (EY) or Aloe vera extract
0h EY 20%
AV 20% a
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(AV), storage during 36 h at 5 ºC (n=5).
36h
EY 20%
AV 20%
45.5 ± 9.0
40.2 ± 17.2b
62.4 ± 5.9
71.4 ± 9.7
Velocity curvilinear (µm/s)
107.1 ± 7.3
107.9 ± 8.9
80.4 ± 20.5
65.2 ± 26.7
Velocity average pathway ( µm /s)
51.7 ± 5.8
47.3 ± 5.6
35.6 ± 9.3
29.6 ± 12.2
Velocity straight line (µm/s)
32.3 ± 5.6
30.1 ± 3.6
20.5 ± 5.4
17.6 ± 7.4
Amplitude lateral head (µm )
6.86 ± 0.4
7.32 ± 0.3
4.68 ± 1.4
4.42 ± 1.9
Beat cross frequency (Hz)
33.7 ± 3.1
35.8 ± 2.7
22.84 ± 6.1
18.7 ± 7.7
Rapid (%) Medium (%) Slow (%) Static (%) a,b
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Subpopulations
60.2 ± 4.7
61.6 ± 1.9
44.8 ± 11.5
33.4 ± 13.7
28.6 ± 3.6
3.0 ± 0.7
21.2 ± 5.6
15.8 ± 6.7
29.6± 7.5
38.6 ± 11.5a
21.5 ± 7.1
20.2 ± 9.8b
22.4 ± 10.6
23.8 ± 4.7
11 ± 2.0
9.7 ± 4.9
10.6 ± 1.8
11.8 ± 3.3
12.7 ± 4.2
10.2 ± 7.0
35.6 ±7.6
b
54.5 ± 9.1
59.7 ± 17.2a
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Linearity (%)
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Straightness (%)
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Total motility (%)
25.8 ± 9.5
Columns with different superscripts differ (P < 0.05) within treatment group (P >0.05).
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Table 2: Values (means ± SEM)* for sperm morphology, viability, osmotic response and plasma membrane integrity in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk or Aloe vera extract in different concentrations. AV 20%
AV 10%
AV 5%
Normal morphology (%)
76.6 ± 1.4
66.9 ± 4.2
67.6 ± 2.7
72.2 ± 3.2
Viability (%)
25.1 ± 2.9
23.3 ± 5.5
21 ± 5.5
20.7 ± 2.4
Osmotic response (%)
34.8 ± 3.7
31.4 ± 5.6
30.8 ± 4.9
28.7 ± 4.8
Membrane integrity (%)
27.5 ± 3.1
27 ± 3.7
27.6 ± 4.2
24.7 ± 3.6
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EY 20%
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Table 3: Values (means ± SEM) for sperm kinetic motility parameters provided by computerized analysis in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk (EY) or Aloe vera (AV) at different concentrations (20, 10, 5%). AV 20%
AV 10%
Total motility (%)
27.1 ± 5.03
46.4 ± 3.9
40.5 ± 7.6
35.7 ± 7
Velocity straight line (µm/s)
24.3 ± 3.6a
20.5 ± 4.7ab
14.9 ± 1.5b
18.4 ± 2.3ab
Velocity average curvilinear (µm/s)
86.8 ± 7.9a
64.7 ± 8.9b
67.3 ± 3.1ab
69 ± 6.3ab
Velocity average pathway (µm/s)
37.8 ± 4.8a
28.6 ± 5.0ab
24.8 ± 1.9b
28.2 ± 2.8ab
Linearity (%)
32.1 ± 2.1ab
33.7 ± 2.4a
26 ± 1.2c
30.5 ± 1.7b
Straightness (%)
64.2 ± 1.9b
71.6 ± 2.6a
60.5 ± 2.1b
64.5 ± 2.8b
Amplitude lateral head (µm)
6.4 ± 0.6
6.6 ± 0.3
5.8 ± 0.3
6.6 ± 0.3
Beat cross frequency (Hz)
34.8 ± 2.0
35.1 ± 1.7
38.7 ± 1.4
37.4 ± 1.4
Rapid (%)
10.1 ± 5.3
6.4 ± 0.7
5.3 ± 1.3
10.2 ± 4.9
Medium (%)
9.7 ± 2.8b
30.4 ± 3.9a
27.7 ± 6.3a
18.2 ± 5.6ab
Slow (%)
7.3 ± 1.9
9.6 ± 1.5
7.5 ± 1.4
7.3 ± 1.7
53.3 ± 4.1b
59.4 ± 8.2
60.3 ± 9.2
Static (%)
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73 ± 6.5
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Superscript low case letters indicate significant differences among treatments (P<0.05).
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Subpopulations
AV 5%
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EY 20%
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Figure 1: Values (means ± SEM) for sperm viability, morphology and osmotic response in collared peccaries (Pecari tajacu) chilled semen (n=5) diluted in Tris plus egg yolk (20%) or Aloe vera extract (20%), storage during 36h at 5
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ºC. (a,b) case letters indicate significant differences for the treatment egg yolk over time and (A,B) case letters indicate significant differences for the treatment Aloe Vera over time (P<0.05).
ACCEPTED MANUSCRIPT Highlights
Aloe vera is an efficient cryoprotectant for peccaries semen chilling and freezing. It can substitute egg yolk in the formulation of Tris extenders.
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We validate the CASA use for evaluate collared peccaries semen.
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We can combine egg yolk and Aloe vera for peccaries semen cryopreservation.
S0093-691X(16)00008-X
DOI:
10.1016/j.theriogenology.2016.01.007
Reference:
THE 13475
To appear in:
Theriogenology
Received Date: 14 January 2015 Revised Date:
4 January 2016
Accepted Date: 4 January 2016
Please cite this article as: Souza A, Lima G, Peixoto G, Silva A, Oliveira M, Silva A, Use of Aloe Verabased extender for chilling and freezing collared peccary (Pecari tajacu) semen, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.01.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Use of Aloe Vera-based extender for chilling and freezing collared peccary (Pecari
2
tajacu) semen
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ALP Souzaa, GL Limaa, GCX Peixotoa, AM Silvaa, MF Oliveiraa, AR Silvaa*
4 5
a
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do Semi-Árido (UFERSA), Mossoró, RN, Brazil
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*Corresponding author. Tel.: 55 84 33178374. E-mail address: [email protected]
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(AR Silva).
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9 Abstract
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Laboratory of Animal Germplasm Conservation - LCGA, Universidade Federal Rural
As an alternative for the conservation of collared peccary semen, this research
12
aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen
13
chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in
14
Tris plus egg yolk—EY (20%) or AV extract (20%)—and chilled at 5 ºC. In both
15
treatments, an adequate semen conservation was achieved and values closer to 40%
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motile sperm with viability and osmotic response ranging from 20 to 40%, and normal
17
morphology of 80% were found after 36 h of storage. Moreover, 12 other ejaculates
18
were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%).
19
Samples were frozen in liquid nitrogen and thawed after one week. After thawing, all
20
the treatments containing EY or AV provided similar values for sperm morphology,
21
viability, osmotic response, membrane integrity, sperm motility, ALH, BCF, and rapid,
22
low, and static subpopulations, but the highest values for STR and the lowest values for
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VCL were found using 20% AV (P < 0.05). In conclusion, we demonstrate that Aloe
24
vera extract at a 20% concentration could be used as an alternative substitute to egg
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yolk in the formulation of Tris extenders for collared peccaries’ semen chilling or
26
freezing.
27 28
Keywords: Tayassu tajacu; sperm chilling; sperm freezing; Aloe vera; cryoprotectant.
30
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29 1. Introduction
31
With the advent of semen conservation, the possibility of germplasm storage
33
enables posterior manipulation and use of assisted techniques such as in vitro
34
fertilization or artificial insemination [1]. Despite this, the decrease in temperature rates
35
during semen chilling and freezing procedures is critical to maintain the sperm quality
36
after rewarming, with the sperm sensitivity varying depending on the species [2].
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To reduce the cryoinjuries, several substances have been used as cryoprotectants,
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and the hen’s egg yolk has showed satisfactory results for both domestic [3, 4, 5] and
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wild species [6, 7, 8]. Egg yolk presents beneficial effects on sperm cryopreservation as
40
a protectant of the plasma membrane and acrosome against temperature-related injury
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[3]. It is believed that the phospholipids, cholesterol, and low-density lipoproteins
42
present in egg yolk may be factors that provide protection to the sperm against cold
43
shock during the freeze–thaw process [4]. However, because it is a product of animal
44
origin, there is the possibility of bacterial contamination [9], which contributes to the
45
difficulty of exchanging semen among different regions or countries [5]. Furthermore,
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egg yolk can interfere with microscopic observations or biochemical assays, as it
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contains granular material of the same size and shape as the sperm [10].
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Several authors search for an alternative medium that is free of animal products
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for semen conservation. In this sense, the Aloe vera appears as an alternative from
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vegetal origin for this purpose, as it constitutes some substances that can act as the
51
conventional cryoprotectants [11]. Along the years, Aloe vera has been used in the
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cosmetic and toiletry industry due to its curative and therapeutic properties [12]. In
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addition, several studies show the positive action of Aloe vera extract on
54
spermatogenesis [13]. The use of Aloe vera extract for an effective semen conservation,
55
however, is restricted to the caprine semen chilling [14] and ovine semen freezing and
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artificial insemination [15]. Therefore, it presents a potential as a good alternative for
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sperm cryoprotection and should be better explored and extrapolated to other species,
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including wild animals.
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The collared peccary (Pecari tajacu) is a wild species whose meat and pelt has
60
great international demand, but the number of individuals in some of its natural habitats,
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such as the Caatinga and Atlantic Forest, is critically decreasing due to poaching, which
62
has necessitated them to be bred under captivity [16]. The development of assisted
63
techniques that make the conservation of collared peccary viable besides the
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implementation of germplasm banks is very important for their multiplication, which
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has generated some studies on its semen conservation. It was demonstrated that their
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semen could be chilled at 17 ºC for only 24 h by using the Beltsville Thawing Solution
67
as extender [17]. At freezing, however, several studies demonstrated that the sperm
68
quality could be efficiently preserved using Tris [18] or coconut water extenders [6] and
69
both plus egg yolk. In order to present an alternative cryoprotectant for the conservation
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of its semen, this research aims at evaluating the use of Aloe vera extract as a
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cryoprotectant for the peccaries’ semen chilling and freezing.
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2. Materials and methods
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The ethics committee of the Universidade Federal Rural do Semi-Árido—
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UFERSA has approved the experimental protocols and the animal care procedures
77
adopted (process no. 23091.0253/114). The reagents used in this study were obtained
78
from Sigma-Aldrich (St. Louis, MO, United States), except when cited.
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2.1. Animals and semen collection
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Samples were obtained from 12 sexually mature male collared peccaries with an
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average (SD) age and weight of 40.7 ± 1.6 mo and 22.5 ± 2.8 kg, respectively. Five of
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the individuals served as semen donors for the semen chilling and freezing, being
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subjected to two electroejaculation sessions on a 30-day interval. The other seven
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animals were used as semen donors for only the semen freezing.
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The animals belonged to the Center of Multiplication of Wild Animals,
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UFERSA, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W).
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The climate is typically semiarid, with an average annual temperature of 27 °C. The
90
animals were isolated from the females at 6 months before the commencement of the
91
study and maintained under a 12-h natural photoperiod. They were maintained outdoors
92
in groups of six in paddocks (20 m to 3 m) with a covered area of (3 to 3 m) and fed
93
sow food and fruits (with water available ad libitum).
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Animals were fasted 12 h before the experiments began. They were then
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physically restrained using a hand net and anesthetized using intravenous administration
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of propofol (Propovan®, Cristalia, Fortaleza, Brazil), which was given as a bolus (5
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mg/kg) [19]. They were kept in lateral recumbency, and the semen was collected with
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an electroejaculator (Autojac®, Neovet, Campinas, SP, Brazil) that was connected to a
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12 V source. The stimulatory cycle comprised 10 stimuli in each voltage, starting from
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5 V, followed by a voltage increase in steps from 1 V to 12 V. Each electrical stimulus
101
lasted 3 s, with intermittent breaks of 2 s. The stimuli cycle was maintained for a 10 min
102
duration from the beginning of the procedure. The electroejaculator probe was 15 x 1.3
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cm, and it was inserted 12 cm into the rectum. Semen was collected into plastic tubes
104
and immediately evaluated [18].
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105 106
2.2. Experimental design
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The execution of the study was divided into two steps. At first, we verified the
109
effect of Aloe vera (AV) extract on the short-term preservation at 5 ºC of collared
110
peccaries’ semen by comparing it with the egg yolk (EY), both at a 20% concentration
111
added to the Tris extender. Afterward, we verified the effect of AV extract at different
112
concentrations (5, 10, and 20%) added to the Tris extender for the collared peccaries’
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semen freezing. In both experiments, we used the Tris-based extender formulation
114
previously described for collared peccaries [18].
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2.3. Extraction of Aloe Vera
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The plant Aloe vera was cultured in a sandy clay soil type at the UFERSA
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campus, located in the northeast of Brazil (Mossoró, RN, Brazil; 5 °10=S, 37 °10=W),
120
under a typical semiarid climate, with an average annual temperature of 27 °C. In order
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to obtain the crude extract of Aloe vera, we extracted the parenchyma of its leaves as a
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colorless gel. It was filtered through a sieve; then, it was packed in a glass container till
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its use.
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2.4. Semen chilling
126 Semen samples that were collected after electroejaculation of 5 individuals were
128
divided into two aliquots. The first was diluted in Tris plus EY (20%), and the other was
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diluted in Tris plus AV extract (20%), reaching a concentration of 100 x 106 sperm/mL.
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Samples were evaluated immediately after dilution (0 h) for sperm total motility,
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viability, osmotic response, and morphology. Then, samples were stored in the water
132
jacket at 27°C and equilibrated for 40 min to reach 15°C in a biological incubator
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(Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to establish a
134
temperature at 5 ºC for 30 min. Samples were rewarmed at 37 ºC and reevaluated every
135
12 h till 36 h.
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2.5.Semen freezing
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Semen was frozen according to a methodology previously developed for
140
peccaries [4]. Samples derived from 12 individuals were divided into 4 aliquots. These
141
were diluted in Tris extender supplemented with EY (20%), as a control group, or AV
142
extract at 5, 10, or 20%. All the dilutions were conducted at room temperature (27 °C),
143
and the samples were then equilibrated for 40 min to reach 15°C in a biological
144
incubator (Quimis, Diadema, SP, Brazil). Furthermore, the incubator was adjusted to
145
establish a temperature at 5 ºC for 30 min. At that point, the sample was added to the
146
extender with 6% glycerol (also at 5ºC), which resulted in a final concentration of 3%
147
glycerol in the extender and 100 x 106 sperm /mL. Finally, the samples were packed in
148
0.25-mL plastic straws (IMV Technologies; L’Aigle, France) that were placed
149
horizontally in an insulated box for 5 min, at 5 cm above the nitrogen (N2) vapors, and
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then plunged into liquid N2 for storage at −196 ºC, following a fast freezing rate at −40
151
ºC/min. After 1 week, frozen straws were thawed by immersion in a water bath at 37°C
152
for 1 min [6]. Samples were evaluated for sperm motility parameters, viability,
153
morphology, osmotic response, and membrane integrity.
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154 2.6. Semen evaluation
156
Fresh ejaculates were evaluated for aspect, color, and volume. The sperm
158
concentration was determined using a Neubauer counting chamber [20]. After
159
electroejaculation, rewarming, and freezing–thawing, samples were evaluated for sperm
160
morphology, viability, and osmotic response. Bengal Rose–stained smears were
161
prepared to evaluate sperm morphology using light microscopy (1000×), counting 200
162
cells per slide [21]. Sperm viability was determined by analyzing a slide stained with
163
bromophenol blue under light microscopy (400×), counting 200 cells per slide [22].
164
Functional integrity of the sperm membrane was verified by evaluating the sperm
165
osmotic response under a hypo-osmotic swelling (HOS) test, using distilled water (0
166
mOsm/L) as the hypo-osmotic solution. A total of 200 sperms were examined, and
167
those with a swollen coiled tail were considered as presenting a functional membrane.
168
[23]. For the evaluation of the plasma membrane integrity in frozen–thawed samples,
169
we used a fluorescent solution that contained fluorophores diacetate 6-carboxy-
170
fluorescein (C-FDA) and propidium iodide (PI). Samples were incubated for 10 min and
171
evaluated by epifluorescence microscopy (Episcopic Fluorescent attachment “EFA”
172
Halogen Lamp Set, Leica, Kista, Sweden). For each sample stained with CFDA/PI, 200
173
sperms were counted and classified as having or not having an intact membrane (based
174
on color) [24].
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2.7. Computer-assisted sperm assessment
177 Sperm motility parameters were analyzed by computer-assisted sperm
179
assessment (IVOS 7.4G, Hamilton-Thorne ResearchTM, Beverly, MA, USA) in fresh,
180
rewarmed, and frozen–thawed semen. The settings of the instrument were validated for
181
collared peccaries semen before analysis, including temperature 37 ºC; 60 frames/s;
182
minimum contrast, 45; straightness threshold, 30%; low-velocity average pathway
183
(VAP) cutoff, 10 µ/s; and medium VAP cutoff, 30 µ/s. Five independent and
184
nonconsecutive microscopic fields were randomly selected and scanned. The following
185
endpoints were analyzed: number of counted cells, total motility (%), VAP (µm/s),
186
velocity straight line (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of
187
lateral head (ALH; µm), beat cross frequency (BCF; Hz), straightness (STR;%), and
188
linearity (LIN;%). According to low VAP cutoff (LVV) and medium VAP cutoff
189
(MVV), the overall sperm population was subdivided into four categories: rapid, with
190
VAP > MVV; medium, with (LVV < VAP
191
the proportion of cells that were not moving [25]. For a trustable evaluation of the
192
sperm tracks, the Edit Tracks Option of the IVOS 7.4G system was used to exclude the
193
debris derived from the extenders. A further dilution in salt solution (1:1) was
194
conducted only if necessary.
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2.8. Statistical analysis
197 198
Results were expressed as mean ± SEM. All analyses were performed using the
199
Statview 5.0 software (SAS Institute Inc., Cary, NC, USA). The data were first
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examined for normality by the Shapiro–Wilk test and for homoscedasticity by the
201
Levene’s test. In view of some data that did not present a normal distribution, an arcsine
202
transformation was conducted for percentage values. The effect of incubation time (0,
203
12, 24, and 36 h) during chilling on sperm parameters was evaluated by variance
204
analysis (ANOVA) for repeated measures. Comparisons among treatments for chilled or
205
frozen–thawed semen were evaluated by ANOVA, followed by the Fisher PLSD test.
206
Statistical significance was set at P < 0.05.
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3. Results
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Fresh ejaculates presented a watery aspect, white color, and a volume of 2 ± 0.4
211
mL with a concentration of 259.2 ± 29.3 x106 sperm/mL. Average values of 97.5 ±
212
1.0% motile sperm, being 83.4± 23% viable and 88.1± 1.5% morphologically normal,
213
with 84.5 ± 3.8% osmotic response were found in fresh samples.
214
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3.1. Semen chilling
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On evaluating the peccary chilled semen during 36 h, no statistical differences
218
were found between samples diluted in Tris plus EY or AV extract for sperm viability,
219
morphology, and osmotic response (Figure 1) at any time. However, we verify a
220
significant linear reduction in the preservation of sperm viability and osmotic response
221
during the experiment for both treatments (P < 0.05) that presented values between 20
222
and 40% at 36 h.
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For CASA evaluation, no samples were rediluted. The kinematic parameters of
224
sperm motility generated by CASA (Table 1) confirmed the similarity between the
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cryoprotectants used for cooling the semen of peccary for 36 hours; however, a
226
significant decrease on the values for total motility was observed in the use of AV after
227
36 h (P < 0.05).
228 3.2 Semen freezing
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After thawing (Table 2), all the treatments containing EY or AV provided
232
similar values for sperm morphology, viability, osmotic response, and membrane
233
integrity.
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Regarding CASA evaluation, the samples diluted in Tris-EY presented large
235
amounts of debris, demanding a long time for its exclusion using the tracks edition
236
option; however, the redilution was only necessary in 3 of the 12 frozen–thawed
237
samples. In contrast, a very low debris formation was observed when using the AV
238
extract in any concentration, where a tracks edition was rarely needed and a redilution
239
unnecessary.
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CASA reveals (Table 3) that all the treatments provided a similar preservation of
241
sperm motility, ALH, BCF, and rapid, low, and static subpopulations, but the highest
242
values for STR and the lowest values for VCL were found using 20% AV (P < 0.05).
243
Some kinetic parameters as STR and LIN were significantly reduced in the groups
244
containing 5 and 10% AV compared to those containing 20% AV (P < 0.05). Moreover,
245
the group containing EG presented values for medium subpopulation lower than those
246
found for 20% AV (P < 0.05).
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247 248 249
4. Discussion
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As previously shown for caprine [14], the AV extract showed an effective
251
cryoprotective effect on peccaries’ semen chilling, providing results similar as the
252
extender containing EY. Chauhan et al. [26] revealed the Aloe vera’s chemistry and
253
reported the presence of more than 200 different biologically active substances,
254
including vitamins, minerals, enzymes, sugars, anthraquinones or phenolic compounds,
255
lignin, saponins, sterols, amino acids, and salicylic acid. In such components, the
256
presence of polysaccharides such as pectin, hemicellulose, glucomannan, and
257
acemannan is highlighted, with the majority being glucose and mannose derivatives
258
[27]. These sugars are known as important energy sources for the sperm [28], and they
259
could have contributed to the peccary sperm’s short-term preservation. Moreover, the
260
presence of substances presenting antioxidant capability such as polyphenols, indoles,
261
and alkaloids was recently detected in the AV extract [29]. This property could add to
262
the prevention of the sperm oxidative damage derived from the hypothermic storage of
263
peccaries’ liquid semen.
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In this study, we demonstrate that the peccary semen could be efficiently stored
265
at 5 ºC during 36 h in the use of a Tris extender supplemented with EY or AV. These
266
results are better than those previously reported for the same species, in which values of
267
only 22% motile sperm were verified after 24 h storage at 17 ºC using the Beltsville
268
Thawing Solution (BTS) [17]. It is known that the use of a storing temperature varying
269
from 15 to 20°C maintains the cellular activity, making sperm more sensitive to toxic
270
metabolic agents [30]. Therefore, the storage of semen in temperatures closer to 4°C, as
271
used in the present study, promotes a reduction in the sperm metabolism, saving its
272
energy reserves, thus contributing to a good preservation of sperm lifespan [30].
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Regarding peccaries’ semen freezing, we verify that extenders containing AV
274
extract provide values for post thawing sperm parameters similar to those previously
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reported for the same species in other studies that used EY plus Tris [18] or coconut
276
water [6] extenders. The use of 20% AV, however, provided the best preservation of
277
STR after thawing. Recently, this parameter, a marker for sperm progression used for
278
describing sperm swimming patterns [31], was positively related to the capability of
279
peccary spermatozoa to interact with swine oocytes [32]. In contrast, the same group,
280
20% AV, provided the lowest values for VCL, which is the motion parameter
281
considered as the main determinant of hyperactivated motility [33]. Therefore, we can
282
assume that the use of 20% AV would be more effective than 20% EY in preventing
283
sperm hyperactivation after freezing–thawing procedures.
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The reduction of AV concentration from 20% to 10 or 5% was not effective in
285
preserving STR or LIN values, and we could not discard the existence of any
286
detrimental activity on the sperm when using AV concentrations higher than 20%. This
287
statement is based on the fact that Gutiérrez et al. [15] tested the AV extract
288
concentrations, varying from 10 to 70%, which were added to coconut water extender
289
for ovine semen freezing. They demonstrate that the best semen preservation is
290
achieved when this extract is used from a 20 to 50% concentration, but if the
291
concentration is increased above 50%, it will present significant detrimental effects on
292
semen quality.
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cryoprotective effect remains unknown. It is known that it contains folic acid and zinc
295
[34] that act as antioxidants, thus improving the semen quality by reducing semen
296
apoptosis [35]. Furthermore, it contains various polysaccharides [27], which, besides
297
serving as an energy source, are known to present some extracellular cryoprotective
298
effects, thus contributing to the sperm membrane stability [36]. In addition, AV extract
299
also contains phenol, saponin, and anthraquinones components that have antiviral and
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antifungal properties [26], and it was recently demonstrated to have antibacterial
301
property against gram-positive and gram-negative pathogens [29]. In this sense, the AV
302
extract could be used as an alternative to EY because EY might contribute to possible
303
risks of contamination, which in turn results in the difficulty of exchanging semen
304
among different regions or countries [10]. Moreover, the amount of debris formation
305
provoked by AV use does not impair the peccary semen evaluation. This statement
306
would be an enormous advantage for the procedure, thus facilitating the manual debris
307
exclusion by using the CASA option for selecting and zooming individual tracks [37].
308
Moreover, AV use would avoid or reduce the need for additional post thawing dilution
309
in isosmotic substances that reduce the debris concentration, thus facilitating the
310
computer analyses [38, 39].
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In conclusion, we demonstrate that the Aloe vera extract could be efficiently used
312
as a substitute for egg yolk in the formulation of Tris extenders for collared peccaries’
313
semen chilling or freezing. Furthermore, the chilling process at 5 ºC provided adequate
314
sperm motility till 36 h by using egg yolk or Aloe vera (20%) in Tris extender. Finally,
315
we suggest the use of Aloe vera extract at 20% concentration in Tris extender for
316
collared peccaries’ semen freezing.
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Acknowledgements
319
The authors thank the Brazilian Council of Scientific Development - CNPq (Process n°
320
552327/2011-5) for financial support; and CEMAS/UFERSA for providing the animals.
321
Ana Liza Paz Souza, Gislayne Christianne Xavier Peixoto, and Andreia Maria Silva
322
were recipients of a grant from CAPES. Moacir Franco de Oliveira and Alexandre
323
Rodrigues Silva were recipients of a grant from CNPq.
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between Neubauer counting chamber and spectrophotometer used for the sperm
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(Pecari tajacu). Arq Bras Med Vet Zootec 2013;65:1257-1260.
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freezing thawing rates on post-thaw survival of dog spermatozoa. Theriogenology
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[32] Campos LB, Souza ALP, Pereira AF, Silva AR. Estimating the fertilizing ability of
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oocytes. Anim Reprod 2015;12:551.
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computer-assisted sperm motion analysis. Hum Reprod 1998;13:2512–2520.
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[35] Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm
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GW, Behne D. Identification of a specific sperm nuclei selenoenzyme necessary for
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Polysaccharide on boar spermatozoa quality following freezing-thawing. Cryobiology
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[37] Mortimer ST, van der Horst G, Mortimer D. The future of computer-aided sperm
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[38] Anel L, Gomes-Alves S, Alvarez M, Borragan S, Anel E, Nicolas M, Martinez-
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Pastor F, Paz P. Effect of basic factors of extender composition on post-thawing quality
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of brown bear electroejaculated spermatozoa. Theriogenology 2010;74:643–651.
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[39] Pillet E, Duchamp G, Batellier F, Beaumal V, Anton M, Desherces S, Schimidt E,
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Magistrini M. Egg yolk plasma can replace egg yolk in stallion freezing extenders.
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Theriogenology 2011;75:105–114.
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Values (means ± SEM) for kinematic parameters of sperm motility in collared
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Table 1:
peccaries (Pecari tajacu) chilled semen diluted in Tris plus egg yolk (EY) or Aloe vera extract
0h EY 20%
AV 20% a
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(AV), storage during 36 h at 5 ºC (n=5).
36h
EY 20%
AV 20%
45.5 ± 9.0
40.2 ± 17.2b
62.4 ± 5.9
71.4 ± 9.7
Velocity curvilinear (µm/s)
107.1 ± 7.3
107.9 ± 8.9
80.4 ± 20.5
65.2 ± 26.7
Velocity average pathway ( µm /s)
51.7 ± 5.8
47.3 ± 5.6
35.6 ± 9.3
29.6 ± 12.2
Velocity straight line (µm/s)
32.3 ± 5.6
30.1 ± 3.6
20.5 ± 5.4
17.6 ± 7.4
Amplitude lateral head (µm )
6.86 ± 0.4
7.32 ± 0.3
4.68 ± 1.4
4.42 ± 1.9
Beat cross frequency (Hz)
33.7 ± 3.1
35.8 ± 2.7
22.84 ± 6.1
18.7 ± 7.7
Rapid (%) Medium (%) Slow (%) Static (%) a,b
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Subpopulations
60.2 ± 4.7
61.6 ± 1.9
44.8 ± 11.5
33.4 ± 13.7
28.6 ± 3.6
3.0 ± 0.7
21.2 ± 5.6
15.8 ± 6.7
29.6± 7.5
38.6 ± 11.5a
21.5 ± 7.1
20.2 ± 9.8b
22.4 ± 10.6
23.8 ± 4.7
11 ± 2.0
9.7 ± 4.9
10.6 ± 1.8
11.8 ± 3.3
12.7 ± 4.2
10.2 ± 7.0
35.6 ±7.6
b
54.5 ± 9.1
59.7 ± 17.2a
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Linearity (%)
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Straightness (%)
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Total motility (%)
25.8 ± 9.5
Columns with different superscripts differ (P < 0.05) within treatment group (P >0.05).
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Table 2: Values (means ± SEM)* for sperm morphology, viability, osmotic response and plasma membrane integrity in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk or Aloe vera extract in different concentrations. AV 20%
AV 10%
AV 5%
Normal morphology (%)
76.6 ± 1.4
66.9 ± 4.2
67.6 ± 2.7
72.2 ± 3.2
Viability (%)
25.1 ± 2.9
23.3 ± 5.5
21 ± 5.5
20.7 ± 2.4
Osmotic response (%)
34.8 ± 3.7
31.4 ± 5.6
30.8 ± 4.9
28.7 ± 4.8
Membrane integrity (%)
27.5 ± 3.1
27 ± 3.7
27.6 ± 4.2
24.7 ± 3.6
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Table 3: Values (means ± SEM) for sperm kinetic motility parameters provided by computerized analysis in collared peccaries (Pecari tajacu) frozen-thawed semen (n=12) diluted in Tris plus egg yolk (EY) or Aloe vera (AV) at different concentrations (20, 10, 5%). AV 20%
AV 10%
Total motility (%)
27.1 ± 5.03
46.4 ± 3.9
40.5 ± 7.6
35.7 ± 7
Velocity straight line (µm/s)
24.3 ± 3.6a
20.5 ± 4.7ab
14.9 ± 1.5b
18.4 ± 2.3ab
Velocity average curvilinear (µm/s)
86.8 ± 7.9a
64.7 ± 8.9b
67.3 ± 3.1ab
69 ± 6.3ab
Velocity average pathway (µm/s)
37.8 ± 4.8a
28.6 ± 5.0ab
24.8 ± 1.9b
28.2 ± 2.8ab
Linearity (%)
32.1 ± 2.1ab
33.7 ± 2.4a
26 ± 1.2c
30.5 ± 1.7b
Straightness (%)
64.2 ± 1.9b
71.6 ± 2.6a
60.5 ± 2.1b
64.5 ± 2.8b
Amplitude lateral head (µm)
6.4 ± 0.6
6.6 ± 0.3
5.8 ± 0.3
6.6 ± 0.3
Beat cross frequency (Hz)
34.8 ± 2.0
35.1 ± 1.7
38.7 ± 1.4
37.4 ± 1.4
Rapid (%)
10.1 ± 5.3
6.4 ± 0.7
5.3 ± 1.3
10.2 ± 4.9
Medium (%)
9.7 ± 2.8b
30.4 ± 3.9a
27.7 ± 6.3a
18.2 ± 5.6ab
Slow (%)
7.3 ± 1.9
9.6 ± 1.5
7.5 ± 1.4
7.3 ± 1.7
53.3 ± 4.1b
59.4 ± 8.2
60.3 ± 9.2
Static (%)
SC
M AN U
73 ± 6.5
EP
Superscript low case letters indicate significant differences among treatments (P<0.05).
AC C
a,b
TE D
Subpopulations
AV 5%
RI PT
EY 20%
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 1: Values (means ± SEM) for sperm viability, morphology and osmotic response in collared peccaries (Pecari tajacu) chilled semen (n=5) diluted in Tris plus egg yolk (20%) or Aloe vera extract (20%), storage during 36h at 5
AC C
ºC. (a,b) case letters indicate significant differences for the treatment egg yolk over time and (A,B) case letters indicate significant differences for the treatment Aloe Vera over time (P<0.05).
ACCEPTED MANUSCRIPT Highlights
Aloe vera is an efficient cryoprotectant for peccaries semen chilling and freezing. It can substitute egg yolk in the formulation of Tris extenders.
AC C
EP
TE D
M AN U
SC
We validate the CASA use for evaluate collared peccaries semen.
RI PT
We can combine egg yolk and Aloe vera for peccaries semen cryopreservation.