DOI: 10.31038/IJVB.2018222

Abstract

The present study aimed at identifying the gastro-intestinal Strongyles of cattle and to evaluate the effect of albendazole on their population dynamics throughout the year. Three cattle farms located in the village of Velambai were used for the study. 175 animals were screened through coprological examination to assess their infection rate (IR) with gastro-intestinal nematodes. 50 Goudali were monitored from April 2015 to March 2016. Of these animals selected, 25 of them received albendazole on day 0 (D0) and constituted the treated group while the other 25 received nothing and stood for the non-treated group. Faeces were examined using the McMaster method to evaluate the efficacy of treatment and to monitor changes in faecal shedding of Strongyles. Faeces from animals were cultured to recover the infective L3 larval stage of Strongyles by the Baermann method. The survey revealed that Strongyles, Strongyloides and Toxocara were the main gastro-intestinal helminths infecting cattle with IRs of 64.5%, 15% and 24.1% respectively. Deworming at the beginning of the rainy season reduced the shedding of helminth eggs (EPG < 400) throughout the season. Percentage reduction in the number of eggs per gram of faeces (EPG) from Strongyles was 68% at day 30. Stool culture revealed the presence of four types of Strongyles with varying abundance depending on the genus (Trichostrongylus, Haemonchus, Cooperia and Oesophagostomum), animal group and month. The genera Trichostrongylus and Haemonchus were dominant throughout the year. Haemonchus spp were significantly sensitive to albendazole between D30 to D90. On D120, albendazole lost its effect with the genus Haemonchus which resulted in the re-infection of the animals, whereas this effect was rather late for the genus Oesophagostomum (from D270).

Key words

Gastro-intestinal helminths, stool culture, deworming, Albendazole, Ngaoundere, Cameroon.

Introduction

Livestock is an important source of income in most developing countries and contributes to food security. In Africa, it contributes upto 10–20% of the gross domestic product (GDP) [1]. However, in the Adamawa Region of Cameroon like in other African regions, this sector is subject to several constraints including diseases [2] for instance gastro-intestinal cattle strongylosis [1]. Strongylosis contributes to emaciation especially at the end of the dry season [3] and leads to production loss. The control of these pathologies in order to improve the individual productivity of cattle is therefore a necessity in a context already marked by the rapid growth of the human population and the increasing demand for animal protein, both in Cameroon and in all developing countries. In Cameroon, particularly in the Adamawa region, deworming has become a common practice by Veterinarians, but most often by breeders themselves and in most cases without seeking for medical advice [1–4]. For almost two decades now, the anti-parasitic pharmaceutical industries have made tremendous advancements in terms of developing new molecules with improved anthelminthic properties, but parasitism still prevails [5]. Just of recent, albendazole was introduced as an essential anthelmintic, but its usage is sometimes abused [6]. In a context where modern breeding is increasingly confronted with problems related to chemical residues (food safety) and the appearance of parasitic resistant strains to the main families of pharmaceuticals around the world, this has exposed the limitations of a systematic deworming and makes the implementation of treatment protocols essential [3–7]. Studies on the efficacy of albendazole on gastrointestinal parasites in calves in the dry season in vina by Sakativa [8] and Sassa et al. [9] in sheep in Mbé in the Adamawa region, revealed the significant impact of gastro-intestinal parasitosis on the productivity of ruminants in the respective areas. But a longitudinal follow-up study on the effect of deworming on the dynamics of gastro-intestinal Strongyles of adult cattle in Vina is lacking. The purpose of this study was to determine the prevalence of cattle helminths and to evaluate the impact of albendazole treatment on faecal egg counts.

Material and Methods

Study zone

This field trial was carried out in Velambai, geographically located between latitude 6° and 8° North and between longitude 11° and 15 ° East. This area is called the ‘Castle of water’ because large number of rivers in the country originates from this locality. Resulting from the emergence of the old crystalline basement, the department of Vina is elevated at an altitude between 1000 and 1300 meters [10]. The high altitude of this region provides a relatively cool climate with temperatures ranging between 22–25°C [11]. The climate is of the Sudanese tropical type with two seasons: the dry season that occurs from November to March, followed by the wet season. The average annual rainfall is 900 mm to 1500 mm. This area is covered by discontinuous vegetation consisting of savanna grasses such as Hyparrheenia, Panicum and Sporobolus. Three cattle farms were randomly selected from the Velambai locality, where 175 animals of the Goudali cattle breed of both study groups received food supplements such as molasses and cotton seed cake (two to three times a week) during the dry season. Coprological assays were carried out on day zero (D0). A study on the prevalence of gastrointestinal parasites was performed on all the 175 selected animals. Faeces was collected from the rectum, stored in a cooler and transported to the Wakwa Agricultural Research Institute for Development (IRAD) Parasitology laboratory for analysis using the McMaster method. The egg per gram of faeces (EPG) ≥ 50 was noted [12–13].

To evaluate the impact of deworming using albendazole on the population dynamics of gastrointestinal strongyles, two groups of cattle were used to monitor the variation of gastro-intestinal Strongyles for 12 months post albendazole administration. 50 cattle of both sexes were selected from the 175 cattle initially selected. These 50 animals were divided into two groups: treated group of 25 cattle including 8 males and 17 females and an untreated group of 25 cattle including 11 males and 14 females. Only the animals in the experimental group (treated group) had undergone deworming with albendazole in bolus (7.5mg / Kg per os).
Fecal samples were taken once a month for 12 months (from April 2015 to March 2016), ie D0, D30, D60, D90, D120, D150, D180, D210 D240, D270, D300 and D330 after the administration of albendazole. Faecal samples of the animals were cultured in a saturated salt solution and larvae were isolated using the Baermann method [14]. Larval identification was carried out using the identification key of [15].

Data Analysis

The One-way analysis of variance (ANOVA) was performed to compare the effect of age, sex, type with infection prevalence. To compare different infection rates of nematodes, the X² test was performed. The EPG averages of the two groups of cattle were compared using the Student t-Test. These different statistics were carried out using the R version 3.2 software. The efficacy of the treatment with albendazole was calculated at day 30 using the method of Presidente [16].

Results

The identification of gastro-intestinal helminths in cattle led to the identification of helminth eggs of veterinary importance i.e. strongyles; Strongyloides papillosus and Toxocara vitulorum. Strongyles were the most common (64.7%) (170.65 ± 7.67). Age and sex were statistically significant (p ≤ 0.05) with infection prevalence. Young animals (1–2 years) were the most infected (85%) and males (76.92%) were more infected than females (60.29%) (Table 1). The prevalence of Toxocara vitulorum was 24.1% (20.3–28). The young recorded a prevalence of 24.28% more infested than adults with a significant difference (p ≤ 0.05) (Table 1). The prevalence of Strongyloides was 12.3% (8.6–16) (Table 1).

Table 1. The prevalence of gastro-intestinal nematodes

^{a, b, c, d}: values with different superscript letters on the same line are significantly different (p < 0.05). µ: mean. sd: standard deviation, EPG: egg per gram

Effect of albendazole on the EPG of gastro-intestinal Strongyles

From D30 to D120 after treatment with albendazole, there was a significant decrease (p < 0.001) in faecal excretion of Strongyles eggs in the treated group (Table II). The EPG’s percentage reduction was 67.15%. In the untreated group, monthly EPG averages were moderate (EPG < 400) throughout the study period. Mean faecal egg shedding variations with respect to Strongyles revealed a peak on D90, but decreased to 68EPG on D330 (Table 2).

Table 2. Effect of albendazole on the EPG of gastro-intestinal Strongyles

*: significatif; **: more significatif; ***: most significatif; µ: mean; sd: standard deviation, NS: no significant difference.

After the coproculture of the L3 nematode larval stages, the following nematodes: Haemonchus spp., Trichostrongylus spp., Cooperia spp., and Oesophagostomum spp were identified at the beginning of the rainy season. The proportion of the nematodes recovered from the different parts of the gastro-intestinal tract was: Abomasum parasites: Trichostrongylus spp. (42%) and Haemonchus spp. (28%), Parasite of the small intestine, of Cooperia spp. (18%), Parasite of the large intestine, Oesophagostomum spp. (12%). These four genera were present throughout the study in all the sampled herds. The variations of the average monthly intensities of the L3 of the Strongyles showed an overall monthly variation of Strongyles. Trichostrongylus sp. was the most common species from April to October with the lowest infection rate (IR) in January. Haemonchus sp. had two peaks: the first one in August and the second higher rate (56%) in January. Cooperia sp peaked in September while Oesophagostomum presented two peaks, the first in October and the second in late January and declined with its lowest rate in March (Figure 1).

Figure 1. Monthly evolution of the L3 stages of the species of the genera of helminthes identified (T, non-treated animals)

The impact of deworming on Trichostrongylus sp. population was remarkable on D90 in the month of July and the percentage L3 shedding significantly declined in January (Figure 2).

Figure 2. Effect of albendazole on Trichostrongylus sp. (E, treated group and T, non-treated group)

Haemonchus in the treated group responded to treatment by recording an L3 reduction from 29% (April-D0) to 12% in May (D30) post albendazole administration (p.a.a). A statistical significant difference (p ≤ 0.05) was observed between the two groups (treated-E and untreated-T) on days 30 and 60 (Figure 3).

Figure 3. Effect of albendazole on Haemonchus sp. (E, treated group and T, non-treated group)

The mean infection prevalence of Cooperia was 12.1% in the treated group and 13.4% in the untreated counterpart. This frequency in the treated group significantly decreased (p ≤ 0.05) at D30 as compared to the untreated group (Figure 4). The occurrence peak of Cooperia sp. in the treated group was observed on D30, > one month after that of the untreated group (Figure 4).

Figure 4. Effect of albendazole on Cooperia sp. (E, treated group and T, non-treated group)

Oesophagostomum sp in the treated group decreased more than that of the untreated group from September (Figure 5). Its peak in the treated group was observed in May. In the untreated group, two L3 occurrence peaks were noticed, the first one in October and the second in January (Figure 5).

Figure 5. Effect of albendazole on Oesophagostomum sp. (E, treated group and T, non-treated group)

Discussion

Prevalence of gastrointestinal nematodes

Of the 175 cattle sampled in three farms in Velambai, 113 (64.5%) shedded Strongyle eggs (mean: 170.65 ± 7.67). This low average EPG observed could be justified by the extreme weather conditions of the dry season which might have limited the survival of the infestive larvae and consequently the parasite load [17], although the effect of larval hypobiosis cannot be ruled-out [18]. This result is close to the  69.57% observed in calves in the Vina by Sakativa [8]. The prevalence of Strongyles was higher in the Velambai 1 farm (76%) than in the Velambai 2 and Velambai 3 farms (70% and 58.58% respectively) with a statistically significant difference between the three farms. These results are due to pasture management. Indeed, the animals of Velambai l and 2 were regularly dewormed which could have reduced the parasite load in these sites. This finding is similar to that of Sassa et al., [9] in small ruminant farms in Vina. Our results show that Toxocara sp. was frequent in the young animals (24.28%). Age and sex had statistically significant (p ≤ 0.05) effects on Toxocara sp occurence. The prevalence of Strongyloides was 12.3%. This prevalence is close to the 9% observed by Ntonifor et al., [4] in the Jakiri area, but far below the 75.5% obtained by Chollet et al., [3] in calves 0–12 months of age in the North and Far North of Cameroon. The low IR here could be due to the average age of the animals (three years six months). Indeed, with Strongyloides there was a strong immunity against this parasite in cattle from the age of 6–9 months [13].

Effect of albendazole on faecal shedding of Strongyles

After one month of treatment, there was a significant decrease (p ≤ 0.05) in the level of faecal egg shedding of Strongyles eggs in cattle receiving albendazole. This result corroborate with those observed in some studies on the resistance of gastro-intestinal Strongyles of ruminants to anthelmintics [19–9]. The percentage response to treated by this group was 68.7%, revealing a form of resistance to albendazole. In fact, in this study, 60% of the animals were adult cattle (more than two years old). These animals could have received several treatments with albendazole, resulting in Strongyles resistance to this molecule [13]. In the untreated group, OPG monthly averages were maintained at a moderate level (EPG < 400) throughout the year. This result confirmed the effect of the rainy season on the variation of Strongyles eggs. Chiejina and Behnke [20] showed that small rains at the end of the dry season resulted in the development of the infective larvae on the pasture. Acquired immunity might have maintained a moderate level of EPG since the group of cattle examined consisted of 60% of animals over two years old. This finding corroborates with that of Elele et al., [21] on cattle in Port Harcourt, Nigeria.

Variation of the larval population of Strongyles

This study revealed multiple infections in cattle in the Adamawa region of Cameroon and this parasitism was similar to that already reported in cattle in many countries in Africa including Burkina Faso [22], Senegal [18] and Cameroon in small ruminants [9]. Similar results with the predominance of Haemonchus and Trichostrongylus genera were also obtained in sheep in Brazil by Klauck et al., [23]. The antagonistic variation between Trichostrongylus sp and Haemonchus could be due to ecological niche competition as the two parasites share the same habitat (abomasum) [24]. On the other hand, this variation on the genera Cooperia (parasite of the small intestine) and Oesophagostomum (parasite of the large intestine) was rather due to an indirect mechanism occuring through the stimulation of the immune reaction of the host or non-specific inflammatory reactions [24]. The effect of deworming on Trichostrongylus sp was observed on the treated group on D90. But this difference was not statistically significant (p > 0, 05). Roeber et al., [25] and Demelash et al., [26] both observed this low sensitivity of Trichostrongylus sp to albendazole in sheep in Australia and cattle in Ethiopia respectively. The percentage of L3 of Trichostrongylus sp was lower at the end of the rainy season in both treated and untreated groups. Pfukenyi and Mukaratirwa [27] also observed low Trichostrongylus sp. L3 levels at the end of the rainy season, which according to these authors could be low due to the transition in climatic factors of the late seasons (rainy and dry). The percentage of L3 Haemonchus sp in the treated group dropped from 29% to 12%, one month (D30) p.a.a. Statistical significant differences (p ≤ 0.05) were observed between treated and untreated groups from D30 to D90. The susceptibility of Haemonchus sp to albendazole was also observed in cattle and equines in Morocco by Zoutien et al., [28] and sheep of Mbé in the Adamawa region of Cameroon [9]. The effect of albendazole on Haemonchus sp from D120 in the treated cattle group was reported to witness some sort of re-infection, especially with the presence of untreated animal faecal material reservoirs on the pasture land. The percentage of the L3 stage of Cooperia was average in both groups throughout the duration of the study (12.1% in the treated group and 13.4% in the untreated group). This may be due to its high resistance to extreme climatic conditions, despite its low fertility [3–29–27]. The effect of treatment with albendazole was significantly ((p ≤ 0.05) different from the untreated group only at day 30. The percentage of L3 of Oesophagostomum sp in the treated group decreased more than that in the untreated group from D90. This could be related to their sensitivity to albendazole that appears to be related to the location of adult worms in the digestive tract of cattle. Indeed, Oesophagostomum is a worm of the large intestine, since albendazole was administered as a 500 mg bolus, this would have required some time for the dissolution in the rumen and thus a late maximum concentration in the large intestine. Holsback et al., [30] also noted this form of resistance of Oesophagostomum in calves in Paraná.

Conclusion

The objective of self-sufficiency in meat products especially in the reduction of the scramble for beef that Cameroon aims at, a new policy on the development of the livestock sector must be established. Our study of gastrointestinal parasites in cattle in Vina demonstrated the validity of the initial hypothesis that gastrointestinal helminths of cattle are predominant in Vina and that the deworming effect of albendazole has an impact on population variation. It showed two peaks in the infestation level during the rainy season with significant intensities, unlike other studies in the area and showed that animals from one to two years were mostly infected. We were able to show the presence of four genera of gastrointestinal Strongyles among which the genera Trichostrongylus and Haemonchus (parasites of the abomasum) were with frequent. Also, it should be noted that deworming at the beginning of the rainy season will keep the animals at a low infection level until the beginning of the dry season. In addition, this study showed that albendazole has a much greater effect on the population of Haemonchus sp and that this effect is late when referring to the population of Oesophagostomum. Treatments could be administered in mid-May (one month after the actual start of the rains) and in late July (second half of the rainy season). Deworming in mid-May will greatly target the genus Trichostrongylus, while doing so at the end of July will greatly target the genus Haemonchus. Finally, this study also revealed high levels of trematode infection that should also be included in control measures. Any proposal for a deworming schedule should be a subject to economic evaluation and should consider the risk of development of resistance to anthelmintics.

Acknowledgements: This work was supported by the vaccine project. We thank the Department of Parasitology and Parasitological Diseases for the material support. We thank IRAD Wakwa for technical and material assistance.

Conflict of interest: Authors declare no conflict of interest

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DOI: 10.31038/IJVB.2018221

Abstract

Cooled and frozen equine semen shows a reduction in fertility, compared to fresh one. In this study, cooled and frozen-thawed equine spermatozoa were compared and analyzed for plasma and acrosomal membrane integrity and mitochondrial membrane potential, combining three fluorescent probes:  H258, CTC, JC-1 with a micro-spectrofluorimetric analysis (Quanticell equipped with a digital system for color images acquisition). Total and progressive motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were measured by CASA system.

We employed an innovative approach to study the reproductive potential of the male gamete subjected to cooling protocols for semen storage. In fact, we evaluated the modifications of equine sperm physiology induced by temperature during cooling and freezing treatments looking at the modifications of different functional sperm characteristics by a simultaneous analysis of different sperm markers with the aim of selecting those that are the most efficient signs for sperm fertility. We identified the mitochondrial membrane potential because it provides useful information on equine sperm quality strictly correlated with fertility. We consider it a useful marker for sperm fertility to be used as a guide to select high-quality semen to be employed in equine breeding farmers.

Keywords

Stallion; Sperm; Cooled semen; frozen semen; Sub-lethal damages

Introduction

Nowadays reproductive technologies are more attractive for the equine industry [1] because of the increasing use of both refrigerated and frozen sexed spermatozoa. Despite the high number of published papers, further knowledge on stallion sperm cryobiology still requires investigations. In the equine species, sperm quality is affected by inter-individual variability, because stallion selection is often based on performance and phenotype more than on sperm quality [2].

Short- and long-term sperm storage is a pre-requisite for the success of artificial insemination. Cooled-stored stallion semen is usually kept at a temperature of about 4-6°C for about 24h. This range of temperature has been evaluated to be efficacious for maintaining motility and consequently fertility near to fresh semen [3] Long-term storage is useful to preserve gametes from high merit animals, to check the health status of semen samples and to share superior genetics among international distributors [4]. Survival of the sperm cell to refrigeration and cryopreservation depends on its shape and size, on its hydration level and on the permeability of its cell membrane. Refrigerated and frozen-thawed semen, used in different artificial insemination programs, show some limits regards viability compared to fresh semen, due to changes in temperature. Temperature variations during a rapid cooling or during cryopreservation of spermatozoa are in fact known to exert deleterious effects on the survival of spermatozoa, resulting in lower conception rates following artificial insemination. Fertility decrease is the result of the reduction of the percentage of motile sperm, or the consequence of morphological abnormalities induced by cryopreservation, as well as of damages of sperm cell plasma membrane [5], that are responsible of the induction of cell death and sub-lethal damages in the surviving population of spermatozoa [6]. Semen evaluation represents a useful tool to investigate male infertility [7] and in many circumstances, a prospective test is desirable to identify an infertile stallion before it embarks on its breeding career [8]. In horse breeding, fertility trials associated to artificial insemination techniques are hampered by the low number of fertilized mares and by the high difficulties in management and insemination [9]. Given the limitations of the standard examination procedures in predicting horse fertility or even in identifying all infertile stallions, many approaches were employed with the hope of defining a relatively straightforward and inexpensive test closely correlated with fertility [10]. The limitation of these approaches is that most tests evaluate only a limited number of those characteristics  necessary for the assessment of sperm fertility rate. For this reason, the combined use of different tests could be more promising to achieve a reliable evaluation of the functional characteristics related to sperm quality [8, 11, 12].

The integrity of sperm membrane is crucial for the maintenance of sperm fertilizing capacity [7] in fact, it is a fundamental requisite for sperm viability and for the success of fertilization. Viable spermatozoa are defined as cells that possess an intact plasma membrane [9]. Several viability assays evaluate the integrity of different plasma membrane compartments by a microscopic or cytofluorimetric approach after cell staining [9, 13]. These techniques, based on the use of viable (SYBR-14) and non-viable propidium iodide (PI) and Hoechst 33258 (H258) dyes allow detecting sperm viability [14].

Acrosome integrity is essential for oocyte fertilization [7, 15], different fluorescent staining such as Pisum sativum agglutinin (PSA) and chlortetracycline (CTC) perform its evaluation. Besides plasma membrane and acrosome integrity, the mitochondrial status plays an important role in determining sperm cell fertility competence because of its relationship with the energetic status of the cell and with motility [9]. The functionality of mitochondria is often studied by rhodamine 123 and MitoTracker fluorochromes but these techniques do not possess the ability to differentiate mitochondria with low or high membrane potential [16]. The lipophilic cationic compound, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) allows for a distinction between spermatozoa with low and high functional mitochondria since this molecule possesses the ability to form aggregates or monomers, each endowed with a different emission spectrum. JC-1 is maintained as a monomer in mitochondria with low membrane potential, emitting green fluorescence, whereas it aggregates in mitochondria with high membrane potential, emitting orange fluorescence. Several authors still employ the sperm chromatin structure assay test (SCSA), based on the metachromatic properties of the acridine orange, to evaluate breaks in the DNA that may have escaped repair during the last steps of spermatogenesis.

Sperm motility is a very important parameter for sperm quality evaluation and it can be assessed using Computer Assisted Sperm Analysis (CASA) system that captures and digitized successive microscopic images [9, 17].

Fluorescent probe association is able to perform a simultaneous evaluation of several sperm cell compartments [18]. It is intuitive that the higher the number of sperm characteristics, the better is the in vitro fertility prognosis [19].

The aim of this study is to evaluate damages, induced by temperature during refrigeration and cryopreservation, on the plasma, acrosomal and mitochondrial membrane of equine sperm cells and to correlate them in order to select the best characteristics useful for a reliable prediction of the fertilizing competence of each semen sample employed in equine breeding.

Materials and methods

Ethical statement

The experiment was approved by the Ethical and Scientific Committee of “Alma Mater Studiorum”, University of Bologna”

Media and Reagents

A modified Tyrode’s medium without bicarbonate was used for incubating sperm in ‘‘non-capacitating’’ conditions. The Tyrode’s medium contained 111 mM NaCl, 3.1 mM KCl, 2 mM CaCl₂, 0.4 mM MgSO₄, 0.3 mM KH₂PO₄, 50 mg kanamycin/ml, 20 mM HEPES, 5 mM glucose, 21.7 mM sodium lactate, 1 mM sodium pyruvate. Stock solutions minus CaCl₂ and pyruvate were prepared, filtered through a 0.2 mm membrane and stored at +4°C [20]. The remaining two ingredients were added 20-24 h before the experiment and the medium maintained under 5% CO₂ in air at +37°C until the beginning of the experiment; pH and osmolality of the medium were maintained at 7.4 and 300 mOsm/kg, respectively.

All chemicals were purchased from Sigma-Aldrich (Milano, Italy) unless otherwise stated.

Semen collection and processing

Semen (eight ejaculates per stallion) was obtained from two individually housed stallions (Standarbred, 6 and 7 years old), routinely used in the artificial insemination programs at AUB-National Institute for Artificial Insemination, located in Cadriano (Bologna, Italy). The management of the stallions and the collection of semen samples were performed in accordance with health and welfare institutional and European regulations.

Ejaculates were collected regularly (three times/week) during the breeding season of 2011, using a Missouri artificial vagina with an in-line filter (Nasco, Fort Atkinson, WI, USA). Gel-free semen volume was measured with a graduated cylinder, semen concentration was determined using a Bürker chamber (Saaringia, Germany) and motion characteristics were estimated with CASA system. Subsequently, the filtered semen was diluted with Kenney’s extender [21] supplemented with a Tyrode medium, to a final concentration of about 20-25 × 10⁶ spermatozoa ⁄ml. One ml aliquot of each fresh semen sample was evaluated by CASA system. After a 10 min incubation period at +37°C, 2 µl of the suspension was loaded onto a pre-warmed (37°C) Leja 20 µm four chamber slide (IMV Technologies, Piacenza, Italy) and seven fields per chamber were analyzed with CASA (HTM IVOS Version 12; IMV Technologies) using the standard setup for equine. The system parameter settings were:  45 frames acquired at 60 frames ⁄s; minimum contrast 70, minimum cell size 4 pixels; lower VAP cut-off 20 µm ⁄s, VAP cut-off for progressive cells 50 µm ⁄s and straightness 75%. Total motility, progressive motility, average path velocity and the number of rapid spermatozoa were recorded [22]. Those ejaculates containing >60% motile spermatozoa after dilution were used for subsequent assessment. Sixteen ejaculates were analyzed. Subsequently, each sample was fractionated in two aliquots:  one was chilled at 4°C, the other one was frozen in 0.5 ml straw. The cooled-semen was placed in commercial styrofoam box and immediately shipped to the laboratory of Department of Emergency and Organs Transplantation (DETO) at the University of Bari, for immediate evaluation, while straws of frozen semen were stored in liquid nitrogen for successive evaluations.

Evaluation of plasma, acrosomal membrane and mitochondrial function

The fluorescent probes:  H258 and CTC allow to evaluate the integrity of the plasmatic and mitochondrial membranes respectively. The first dye enters the cell and stains the nucleus when plasma membrane is damaged while the second enters the cell when the acrosomal membrane is damaged. Mitochondrial status was assessed by the lipophilic cationic JC-1 that differentiates mitochondria with low or high membrane potential by colors.

A stock solution of H258 was prepared to dissolve 10 mg in 100 µl distilled water. It was wrapped in foil and kept at 4°C. For use, 1 µl of the stock solution was diluted with 10 ml protein-free medium (PBS) and kept at +4°C. The fixative was made by mixing 1: 1 (v/v) 25% glutaraldehyde and 1 M Tris, pH 7.4 [23].

The CTC solution was made of 0.75 mM CTC and 5 mM L-cysteine in chilled CTC buffer containing 20 mM Tris and 130 mM NaCl, pH 7.8 [24].

The stock solution of 1.53 mM JC-1 in DMSO was prepared prior to use [16]. Different fluorescent probes with characteristics of excitation/emission were used simultaneously.

For each sample, aliquots of refrigerated and cryopreserved semen, pre-warmed at 37°C and to a final concentration of about 20×10⁶ spermatozoa/ml, were suspended in 1 ml of Tyrode’s medium and JC-1 solution (2 µM final concentration) was added. Cells were incubated for 15 min at 37°C, then washed in PBS and incubated again for 2 min with 2 µl H258 solution. Unbound dye was removed centrifuging (900xg for 5 min) the stained suspension on 500 µl of 2% (w/v) polyvinylpyrrolidone in PBS.

The supernatant was discarded and the pellet re-suspended in 45 µl Tyrode’s medium. To each sample, 45 µl of the CTC solution and 4 ml of fixative solution were added and mixed. Few droplets of this suspension containing fixed and stained spermatozoa were placed between two coverslips and visualized by a Quanticell micro spectrofluorimetric system (VisiTech International, Sunderland, UK), consisting of a video digital imaging apparatus for the acquisition of color images (Nikon Instruments).

Plasma membrane integrity was evaluated using a 346-460 nm wavelength excitation/emission filters. Acrosomal membrane integrity was assessed by CTC at the 392-536 nm wavelength (excitation/emission). In mitochondria with high membrane potential, JC-1 forms multimeric aggregates and when excited at 480 nm, it emits light at 590 nm (high orange), on the contrary within mitochondria with low membrane potential, JC-1 forms monomers emitting at 525-530 nm (green) when excited at 488 nm wavelength.

For each ejaculate, about two hundred spermatozoa were analyzed and classified according to their florescence emission.

Sperm motility

Sperm kinetic was evaluated using a Sperm Analyzer (CASA-system; HTM-IVOS, Version 12.3, Hamilton-Thorne, Biosciences, MA, USA). Each ejaculate was analyzed for the cooled and frozen aliquots. Briefly, a 2 µl drop was recovered and placed in a Leja 4 analysis chamber, 20 micron depth (Leja Products B.V., The Netherlands). The analysis was performed at 37°C. The following motility characteristics were measured for each sample:  percentage of motile spermatozoa; percentage of spermatozoa with a progressive motility; average path velocity (VAP):  the velocity of the smoothed cell path (µm/s); straight line velocity (VSL):  the average velocity measured in a straight line from the beginning to the end of the track (µm/s); curvilinear velocity (VCL):  the average velocity measured over the actual point to point track followed by the cell (µm/s); amplitude of lateral head displacement (ALH); beat cross frequency (BCF); linearity (LIN); straightness (STR) and the percentage of rapid, medium, slow and static spermatozoa.

It has been necessary to set up the software as previously indicated, to clearly identify all spermatozoa, moreover to be sure that all sperm trajectories were correctly analyzed by CASA, the playback mode was also activated. Seven microscopic fields, selected randomly, were scanned.

Statistical analysis

The analysis of variance (ANOVA) was used to evaluate all recorded parameters. Differences were considered significant when P< 0.05. Linear regression and correlations among variables were also calculated.

Results

Evaluation of plasma, acrosomal membrane and mitochondrial function

At first, in each sample (cooled and frozen), the integrity of both the plasmatic and acrosomal membrane were evaluated recording the percentage of male gametes with intact or damaged membrane, moreover the percentage of spermatozoa with high or low mitochondrial potential was also recorded (Figure 1).

Figure 1. Photomicrography of equine spermatozoa. A Spermatozoa stained with H258 (Hoechst 33258), B Spermatozoa stained with CTC (chlortetracycline), C Spermatozoa stained with JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl carbocyanine iodide).

NIS-Elements F 3.0 (Nikon Instrument) software was employed to evaluate simultaneously all fluorescent probes. The results were used to classify spermatozoa in eight classes (Table 1), according to the morpho-functional state of plasmatic and acrosomal membranes and to mitochondrial function (Figure 2).

Table 1. Classification of equine sperm cells according to fluorescence emitted by H258, CTC and JC-1 probes.

 ^aH258 positive (+) = blue stained nucleus. ^bCTC positive (+) = green acrosome region. ^cJC-1 positive (+) = red in mid-piece region; negative (-) = green in mid-piece region.

Figure 2. Photomicrography of equine spermatozoa stained with H258,CTC and JC-1. A) Intact plasma membrane, intact acrosome and high mitochondrial potential, B) Intact plasma membrane, intact acrosome and low mitochondrial potential, C) Intact plasma membrane, damaged acrosome and high mitochondrial potential, D) Intact plasma membrane, damaged acrosome and low mitochondrial potential, E) Damaged plasma membrane, intact acrosome and high mitochondrial potential, F) Damaged plasma membrane, intact acrosome and low mitochondrial potential, G) Damaged plasma membrane, damaged acrosome and high mitochondrial potential, H) Damaged plasma membrane, damaged acrosome and
low mitochondrial potential.

The analysis revealed that both cooled and frozen-thawed samples had similar amount of spermatozoa with intact or damaged plasma membrane, also the percentage of cells with intact and damaged acrosomal membrane was not statistically different; on the contrary a dramatic decrease in the percentage of spermatozoa with high mitochondrial potential was observed in frozen samples (Table 2).

Table 2. Recovery rate:  cooled versus frozen semen

Recovery rate (%) of spermatozoa showing different morphology of the acrosome and plasma membranes and mitochondrial potential in cooled and frozen semen.

Compared to cooled semen, in cryopreserved samples we recorded a reduction in the number of spermatozoa with intact plasma membrane, intact acrosome and high mitochondrial potential (6%); spermatozoa with intact plasma membrane, damaged acrosome and high mitochondrial potential were also lower (16%), as well as those with damaged plasma membrane, intact acrosome and high mitochondrial potential (1%). Moreover, also the number of spermatozoa with the damaged plasma membrane, intact acrosome and low mitochondrial potential were slightly reduced in frozen samples. While differences described above were not statistically significant, the reduction, caused by cryopreservation, in the class of intact plasma membrane, damaged acrosome and high mitochondrial potential spermatozoa was statistically significant (P< 0.001) as well as in the class of damaged plasma membrane, intact acrosome and low mitochondrial potential spermatozoa (P< 0.05) (Table 3).

Table 3. Cooled versus frozen semen.

Recovery rate of spermatozoa (%) for each of the eight morphological cell classes.

Cryopreservation led to an increase of spermatozoa with intact plasma membrane, intact acrosome and low mitochondrial potential spermatozoa (9%), of intact plasma membrane, damaged acrosome and low mitochondrial potential spermatozoa (13%), of damaged plasma membrane, damaged acrosome and low mitochondrial potential spermatozoa (9%), compared to refrigeration. These increases due to cryopreservation were statistically significant for intact plasma membrane, intact acrosome and low mitochondrial potential spermatozoa (P<0.05), for intact plasma membrane, damaged acrosome and low mitochondrial potential spermatozoa (P< 0.05) and for damaged plasma membrane, damaged acrosome and low mitochondrial potential spermatozoa (P< 0.05).

Sperm motility

Motility analysis was also conducted on each ejaculate of fresh semen; the recorded mean values of total and progressive motility were 79.06% ± 3.8 and 63.87% ± 2.16, respectively.

Sperm motility values of cooled and frozen-thawed semen are shown in Table 4. Semen cryopreservation affected sperm motility. Refrigerated semen displayed higher total and progressive motility than frozen-thawed semen (P< 0.001). The estimated reduction in the percentage of total and progressive motile spermatozoa related to cryopreservation was 20.33% and 19.57%, respectively.

Table 4. Motility parameters:  cooled versus frozen semen.

Motility parameters of equine cooled or frozen sperm samples:  VAP (average path velocity); VSL (straight line velocity); VCL (curvilinear velocity); ALH (amplitude of lateral head displacement); BCF (beat cross frequency); STR (straightness); LIN (linearity). * P < 0.05, ** P < 0.001.

Evaluated VAP and VCL were lower in frozen-thawed semen than in refrigerated one (P< 0.001).

The VSL of refrigerated spermatozoa did not differ significantly from that of cryopreserved sperm.

The ALH was higher in cooled than in frozen-thawed semen (P<0.001). The BCF of cooled spermatozoa did not differ significantly from that of frozen sperm cells.

Cooled semen displayed a higher percentage of rapid spermatozoa (P<0.001) while a 28.82% reduction was induced by cryopreservation. The percentage of medium spermatozoa was similar in cooled and frozen semen. Slow and static spermatozoa were higher in frozen-thawed than in cooled semen (P<0.05) the percentage increase, probably due to the cryopreservation process, was 6.83% and 21.34%, respectively.

Correlations

Significant correlations were observed between mitochondrial energy level and different sperm membrane conditions. In cooled semen a high correlation between high mitochondrial potential and intact plasma membrane (r=0.91), high mitochondrial potential and intact acrosome (r=0.93), low mitochondrial potential and damage acrosome (r=0.90), were found. In frozen semen, a high correlation between low mitochondrial potential and damaged plasma membrane (r=0.87), low mitochondrial potential and damaged acrosome (r=0.92) were found. On the contrary, no correlations between mitochondrial membrane potential and different motility characteristics were evidenced.

Discussion

Severe changes in temperature are a common feature of semen preservation protocols but are not a biological phenomenon to which the sperm cell is adapted [25]. Temperature transitions associated with semen fast chilling or freezing are in fact well known for their production of deleterious effects on sperm survival and consequently, lower conception rates following artificial insemination [5].

The reduction in fertilizing ability has typically been attributed to a reduced rate of sperm motility and to the induction of morphological abnormalities [5]. Damages to the plasma membrane, to the acrosomal membrane and to the mitochondrial function of spermatozoa undergoing cooling and/or freezing depend on changes of temperatures and osmolarity, which cause morphological alterations in the organization and composition of proteins and lipids of the sperm surface [26].

Sperm quality related to functional characteristics has been investigated by the association of different fluorescent probes in ram [27], bull [28] and stallion [13, 16, 18].

In this study we validated the association of three fluorescent probes H258, CTC and JC-1 to evaluate the state of plasma and acrosomal membranes, as well as the mitochondrial function both in cooled and frozen equine sperm, using the inverted fluorescent microscopy highlighting the prominent role of the mitochondrion as marker for equine sperm quality.

We found both in cooled and frozen equine semen samples a higher number of spermatozoa with intact plasma membranes compared to those with destroyed membranes, this result is in line with the observations reported by Vasconcelos et al., [29] and by De Leeuw et al., [30].

It is reasonable to attribute the poor fertility of frozen semen to ultrastructural modifications of the cell membranes and to phase transitions occurring during cooling and rewarming. It seems reasonable that the reorganization of sperm membrane bilayer alters the interactions among lipids and between lipids and proteins required for normal membrane functions, determining a re-modelling of the membrane components [31, 32] and the decreased amount of polyunsaturated fatty acids and cholesterol [33]; [34]. Plasma membrane disruption due to cooling or freezing determines the loss of cations and enzymes from the sperm cells, providing some explanation for the reduction in sperm motility and metabolic activity observed in cold-shock spermatozoa [35]. Cold shock also destroys the selective permeability of sperm membranes to calcium, thus leading to its excessive intracellular accumulation, and consequently, to a reduction in motility and cell necrosis. These processes result in an advanced stage of sperm maturation characterized by capacitation, hyperactivation and acrosome reaction [35].

For what concerns the acrosome membrane, in the present study cooled spermatozoa displaying their integrity, were more numerous than those with a damaged acrosome membrane. As to mitochondrial membrane potential, it is a sensitive indicator of the functional status of the organelle; in the present study, cooled spermatozoa with intact plasma membrane showed a high mitochondrial potential. Whereas a high percentage of spermatozoa with an intact plasma membrane and low mitochondrial potential was observed among cryopreserved ones, suggesting that cryopreservation may result in a significant loss in mitochondrial potential.

Cryopreservation amplified the mitochondrial damage compared to cooling, probably because of the block in ATP synthesis and consequently in the reduction in the activity of ATP-dependent pumps [36], a tendency to make the plasma membrane unstable. This may lead to an increase in intracellular Na⁺ and a reduction in intracellular K⁺, with reduced Ca²⁺ efflux due to inhibition of the Ca²⁺ ATP-ase pumps, increased leakage of Ca²⁺ across the membrane due to the instability caused by removal of cholesterol, and/or increased Ca²⁺ influx due to the activation of unidentified channels [37]. Changes associated with this process include an increase of capacitated and acrosome-reacted spermatozoa, as reported by Felix [37]. These results are in agreement with those reported by Albrizio et al., [38]; they showed that cryopreservation increases the percentage of capacitated and acrosome reacted spermatozoa, increase the intracellular Ca²⁺ concentration  and leads to a higher functional response of L-type Ca²⁺-channels to both the agonist (Bay K-8644) and antagonist (Nifedipine). Moreover, cryopreservation was found to determine the activation of lytic enzymes and to increase mitochondrial permeability, leading to cell death [10]. This is in accordance with the results of the present study, in which the percentage of spermatozoa with damaged acrosome membrane and low mitochondrial potential is higher in frozen than in cooled ones.

It has been suggested that plasma membranes and mitochondria in bovine sperm are both affected by cryopreservation [39]. In accordance with the above-mentioned study, we found that plasma membrane and mitochondrial membrane were equally vulnerable to the freezing and thawing process, because of their high thermolability [40]. Moreover, in this study, plasma membrane and mitochondrial membrane conditions are highly correlated demonstrating that these structures depend on each other, as reported by Celeghini et al., [7]. The mitochondrial plasma membrane is a good predictor to determine and assess plasma membrane and acrosome membrane damages because of thermic treatment. Besides structural sperm characteristics, conflicting opinions concerning the relationship between motility and fertility in the stallion exist [41, 42] although sperm cell motility and the quality of motility are still the most reliable indicators of sperm viability. In this study, total motility recorded in cooled semen was in agreement with similar data reported by Love et al.,[13] and Heckenbichler et al., [43], whereas, the same parameter found in frozen semen was similar to that found by Wrench et al., [44] and Salazar et al., [45]. The progressive motility found in cooled semen was similar to that reported by Cocchia et al., [46] and Heckenbicher et al., [43], whereas the same parameter found in frozen semen was similar to that found by Wrench et al., [44]. It is well known the functional relationship between the mitochondrion and the cellular apparatus that drives sperm movements; in fact mitochondria are well known for producing energy in the form of ATP and sperm motility depends upon the energy produced by the oxidative phosphorylation. Mitochondria are a major source of Reactive Oxygen Species (ROS) and appear to be the cellular structures most susceptible to damages during cooling and cryopreservation [47], leading to a loss of motility in frozen semen.

As to VCL, VAP, VSL and ALH parameters, the results found in cooled semen were in agreement with those of Aurich et al., [48], whereas those found in frozen semen were similar to the results of Salazar et al., [45].

This study demonstrates that the quality of motility is different in cooled and frozen semen, in fact, cooled samples showed higher VAP and VCL values than frozen ones. These results suggest that motile cells, that are numerically higher in cooled than in frozen semen, are also characterized by higher speed as deduced by their higher VAP and VCL, on the contrary, linear characteristic of sperm motility is not affected by temperature reduction. We found that ALH values were higher in refrigerated semen than in the cryopreserved one. In general, higher ALH values are unwanted, because they can interfere with cell progression, as demonstrated by [18], which stand for reduced sperm quality. However until now a cutoff value for ALH has not been defined so that is not clear the influence of this parameter on the progressive motility and on the ability of the spermatozoon to go forward into the female reproductive tract [7].

As to motility characteristics, other authors found opposite results for refrigerated and cryopreserved semen [42]; [49]; [50]. This could be only an apparent discordance considering that many factors may have a role such as season of semen recovery, physiological differences among stallions, frequency in collecting semen, different extenders for semen dilutions, different settings of the CASA system (threshold setting, specimen concentration, video digitalization rate) [9, 17, 51-53].

We, as several authors working on other animal species, didn’t find a direct correlation between mitochondrial function and a specific kinetic parameter, probably because equine sperm motility is modulated by different factors not only by mitochondria [14, 23].

In conclusion, our study, reporting the simultaneous evaluation of different functional characteristics of equine spermatozoa subjected to temperature changes, increases the information on semen quality helping the selection of those characteristics more correlated with semen fertility potential. In particular, we demonstrated that mitochondria are good indicators of sperm survival and can be used to discriminate between good and poor semen quality. Moreover, they are strictly correlated to the state of cellular membrane, therefore, a proper evaluation of mitochondria sounds necessary when analyzing sperm samples. Standing on the present study, mitochondrial membrane potential provides useful information on equine sperm quality so that it may be exploitable as a marker of sperm fertility; moreover, the high mitochondrial sensitivity to temperature variations observed in this study highlights the importance of developing new strategies to protect the functionality of this organelle to preserve sperm cell physiology.

Disclosure

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

This paper was concepted by GM Lacalandra, G. Mari and A. Zarrilli; written by M. Albrizio, G. Mari, GM Lacalandra and A. Zarrilli. Data were acquired by AC Guaricci, A. Moramarco and E. Micera; B. Mislei, and G. Rizzato analysed and interpreted the data. Albrizio revised the manuscript and cared the journal submission.

All authors approved the final article.

Acknowledgements: This work was supported by Italian University funds

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