Environmental Factors Related to a Semiarid Climate Influence the Freezability of Sperm from Collared Peccaries ( Pecari tajacu Linnaeus,1758)

Abstract

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male collared peccaries was collected by electroejaculation during the peaks of the dry and rainy periods while rainfall indices, air temperatures, relative humidity levels, and wind speeds were measured. The number, motility, morphology, osmotic response, and membrane integrity of sperm in the collected ejaculates were assessed. Samples were then frozen in liquid nitrogen, thawed, and reassessed. The rainfall index of the rainy period (73.2 mm) was significantly higher than that of the dry period (13.6 mm) and the relative humidity was significantly higher during the rainy period (74.6%) than it was during the dry period (66.8%). Air temperature and wind speed did not differ between the two periods. Characteristics of sperm in the fresh samples were not affected by environmental parameters. In contrast, computerized analysis revealed that sperm in samples frozen during the rainy period exhibited better post-thaw membrane integrity (28.6 ± 6%), motility (29.5 ± 7.7%), and rapid sperm population (13.7 ± 6.2%) than did sperm in samples frozen during the dry period (23.4 ± 3% membrane integrity, 14.6 ± 4.1% motility, and 4.1 ± 1.2% rapid sperm; p < 0.05). Other characteristics of the frozen/thawed sperm did not differ depending on the period in which they were collected. We demonstrated that environmental parameters did not affect the quality of fresh sperm, but could influence the freezability of sperm collected from collared peccaries raised under a semiarid climate.

Introduction

The collared peccary (Pecari tajacu) is a species of wild pig native to the Americas.¹ Although the International Union for the Conservation of Nature classifies the collared peccary as a least concern species,² poaching and deforestation are reducing populations in some biomes.³ One such biome is the Caatinga, which is characterized by a semiarid climate, irregular rainfall, and high average annual temperatures.⁴

Seasonal changes in temperature can influence the metabolism⁵ and activities of peccaries.⁶ Thus, temperature and other environmental factors might also influence the reproductive traits of peccaries, such as sperm quality, as has been shown to occur for other mammals living in the Caatinga.^7,8 Environmental factors have also been shown to affect the freezability of the sperm of some wild species, such as the Iberian ibex (Capra pyrenaica).⁹ However, how the environment influences the freezability of sperm from collared peccaries remains unknown.

To gather information that will contribute to the establishment of an efficient biobank, the current study aimed to evaluate the influence of certain environmental parameters of Caatinga's semiarid climate on the characteristics of fresh and frozen/thawed sperm collected from collared peccaries.

Materials and Methods

Animals

The Ethics Committee of Animal Use of the Federal Rural University of Semiarid Region (CEUA/UFERSA; no. 23091.008820/2016-03) approved this study. Sperm samples were taken from 11 male collared peccaries that were 40.7 ± 1.6 months old and weighed 22.5 ± 2.8 kg. The animals belonged to the Center of Multiplication of Wild Animals (CEMAS/UFERSA), located in Mossoró, RN, Brazil (5°10 = S, 37°10 = W). The climate there is typically semiarid, with an average annual temperature of 27°C. The animals were maintained under a 12-hour natural photoperiod in groups of six in paddocks (20 × 3 m) with a covered area of 3 × 3 m. Animals were fed pig food and fruits and were provided with water ad libitum.

Evaluation of environmental parameters

The experiment presented here was conducted during the peaks of the dry period (October–December, 2015) and rainy period (March–May, 2016). The rainfall index (mm) for each period was measured automatically by a station of the National Meteorological Institute.¹⁰ Air temperature, relative humidity, and wind speed were measured with a thermo-hygrometer-anemometer (INSTRUTERM-HT300; São Paulo, Brazil).¹¹ These parameters were measured every hour, from 6 a.m. to 6 p.m., on days when sperm was collected.

Sperm collection and evaluation

During the peaks of the two climatic periods, semen was collected from each male once per month for 3 months. For sperm collection, animals were anesthetized with propofol (5 mg/kg IV; Propovan, Cristalia, Fortaleza, Brazil) after fasting for 12 hours. Semen was then collected into plastic graduated tubes by electroejaculation as previously described for the species.¹² Sperm concentration was measured with a Neubauer chamber and the total number of sperm was calculated.¹³ Sperm morphology was assessed by analyzing a smear of 100 cells stained with bromophenol blue under light microscopy (×1000).¹³ Sperm membrane functionality was determined by measuring response to hypoosmotic shock induced with distilled water (0 mOsm/L).¹⁴ Membrane integrity was assessed via fluorescence microscopy using carboxyfluorescein diacetate and propidium iodide as probes.¹⁵ Sperm kinetic motility patterns were analyzed by a computer-aided semen analysis (CASA) system (IVOS 7.4G; Hamilton-Thorne Research, Beverly, MA) using configurations determined for the species.¹⁵

Sperm freezing and thawing

Semen samples were diluted at 27°C in a Tris extender supplemented with 20% Aloe vera, incubated for 40 minutes to reach 15°C, and then incubated again for 30 minutes at 5°C. Glycerol was then added to the samples to a final concentration of 3% (resulting in samples containing 100 × 10⁶ sperm/mL) and the samples were packed in 0.25-mL straws. The samples were frozen via exposure to nitrogen (LN₂) vapors for 5 minutes and then plunged into LN₂ for storage. After 2 weeks, the samples were thawed by bathing in water at 37°C for 1 minute¹⁵ and the sperm was analyzed in the same manner as was the fresh sperm.¹⁵

Statistical analysis

Data (mean ± standard error) were tested for normality with the Shapiro–Wilk test and asymmetries and kurtosis were assessed with the univariate procedure provided by the Statistical Analysis System application (SAS 8.0; SAS Institute, Inc., Cary, NC). When necessary, data were transformed by log (x + 1) or arc sine (√(x/100)). Effects of individuals and periods (dry and rainy) on semen parameters were evaluated by an analysis of variance using a general linear model (Proc GLM; SAS Institute, Inc.). Comparison of means was conducted by Student's t tests (p ≤ 0.05). Efficiency of semen collections during dry and rainy periods was evaluated by the chi-squared test (p ≤ 0.05).

Results

The total rainfall index measured during the peak of the dry period (13.6 mm) was significantly lower than that measured during the rainy period (73.2 mm) (p < 0.05). Values for environmental parameters that were measured during the experiment are reported in Table 1. The relative humidity during the rainy period was higher than that during the dry period (p < 0.05).

Table 1.

Mean Values (Minimum and Maximum) for the Environmental Parameters Verified During Dry (October, November, and December 2015) and Rainy (March, April, and May 2016) Periods in Caatinga's Semiarid Climate

	Wind velocity (m/s)		Air temperature (°C)		Relative humidity (%)
Period	Minimum	Maximum	Minimum	Maximum	Minimum	Maximum
Dry	0.4	4	27.1	36	34	66.8^b
Rain	0.06	2.9	26.7	37.2	42.2	74.6^a

a,b

Means without common superscript letters differ within a row (p < 0.05).

There was no effect of climatic period on the efficiency for semen collection (p > 0.05). All the males provided at least one ejaculate per period. During the dry period, 14 semen samples were obtained from 33 attempts (14/33; 42.4%); three males provided two ejaculates while the other eight males ejaculated only once. During the rainy period, from the 33 attempts conducted, 11 ejaculates were collected (11/33; 33.3%), being one ejaculate provided per male.

Fresh sperm collected during the dry period was not found to differ from fresh sperm collected during the rainy period (Tables 2 and 3). In contrast, semen cryopreserved during rainy period presented more intact membranes than that frozen during the dry period (Table 2). Moreover, CASA (Table 3) revealed that cryopreserved sperm collected during the rainy period was more motile and rapid moving than the cryopreserved sperm collected during the dry period (p < 0.05).

Table 2.

Values (Mean ± Standard Error of the Mean) for Semen Characteristics of Fresh and Frozen/Thawed Samples Derived from Collared Peccaries (Pecari tajacu) Bred Under Semiarid Conditions, and Collected by Electroejaculation During the Dry (October, November, and December 2015; n = 14) and Rainy (March, April, and May 2016; n = 11) Periods of Caatinga's Semiarid Climate

	Fresh semen		Thawed semen
Sperm variables	Dry period	Rainy period	Dry period	Rainy period
Sperm/ejaculate (×10⁶)	1336.5 ± 296.8	1888.6 ± 590.5	—	—
Membrane integrity (%)	84.4 ± 2.4	74.4 ± 7.6	23.4 ± 3^b	28.6 ± 6^a
Osmotic response (%)	79 ± 4.5	78.6 ± 6.3	23.6 ± 3	24.3 ± 4.3
Normal morphology (%)	86.9 ± 2.5	86.8 ± 3.2	76.8 ± 1.2	79 ± 2.1

a,b

Superscripts mean differences between semen collected in different periods.

Table 3.

Values (Mean ± Standard Error of the Mean) for Kinetic Parameters of Sperm Motility of Fresh and Thawed Semen Derived from Collared Peccaries (Pecari tajacu) Bred Under Caatinga's Semiarid Climate During Dry (October, November, and December 2015; n = 14) and Rainy (March, April, and May 2016; n = 11) Periods

	Fresh semen		Thawed semen
Sperm parameters	Dry period	Rainy period	Dry season	Rainy period
Total motility (%)	78.6 ± 4.4	85.7 ± 3.3	14.6 ± 4.1^b	29.5 ± 7.7^a
Velocity average pathway (mm/s)	42.6 ± 2.6	49.5 ± 3.5	22.5 ± 1.6	24.3 ± 1.4
Velocity straight line (μm/s)	27.1 ± 2.2	32.6 ± 2.8	13.5 ± 1.1	16.2 ± 1.4
Velocity curvilinear (μm/s)	100 ± 4.4	106.7 ± 5.9	58.9 ± 4	59.2 ± 3
Amplitude lateral head (μm)	5.9 ± 0.2	6.2 ± 0.2	5.2 ± 0.5	5.5 ± 0.7
Beat cross frequency (Hz)	40.1 ± 1.2	36.8 ± 1.2	38.2 ± 0.8	35.8 ± 1.7
Straightness (%)	62.7 ± 2	64.4 ± 1.3	64.8 ± 1.9	65.7 ± 3.3
Linearity (%)	30 ± 1.6	31.7 ± 1.3	31.5 ± 1.4^b	33.2 ± 3.7^a
Subpopulations
Rapid (%)	54.7 ± 5.4	68.1 ± 6	4.1 ± 1.2^b	13.7 ± 6.2^a
Medium (%)	21.1 ± 2.5	17.8 ± 3.2	14.2 ± 4.2	15.8 ± 3
Slow (%)	5.7 ± 0.7	3.8 ± 0.6	6.6 ± 1.4	6.4 ± 1.2
Static (%)	18.5 ± 4.2	10.3 ± 2.9	75.1 ± 6.3	64.1 ± 8

a,b

Superscripts mean differences between semen collected in different periods.

Discussion

There is growing concern that genetic materials from valuable wild species should be safeguarded in biobanks. Doing so will require more knowledge about basic aspects of reproductive biology and cryobiology.¹⁶ Because of the ecological and economic importance of collared peccaries, recent studies have focused on developing protocols for preserving their reproductive¹⁵ and somatic cells.¹⁷ In addition to helping to preserve collared peccaries, advances in these protocols could be extrapolated to help preserve other related species, such as the vulnerable white-lipped peccary (Tayassu pecari Link, 1795) and the endangered Chacoan peccary (Catagonus wagneri Rusconi, 1930).² The results of the present study contribute to the development of a protocol for efficient biobanking by demonstrating that sperm from peccaries raised under semiarid conditions would be cryopreserved better if collected during the rainy period, even though the quality of their fresh sperm is not affected by environmental factors.

We demonstrated that it is possible to obtain semen from peccaries raised in a semiarid region independently from the climatic period. However, not all individuals provided samples in all the attempts for semen collection. This is probably a consequence of an individual variation related to the electroejaculatory procedure observed in the species, as previously reported by Peixoto et al.¹³

Differences in the rainfall indexes and relative humidity levels of the rainy and dry periods did not affect the number of sperms produced by peccaries. This finding contrasts with reports that goats raised in the same semiarid region produced more sperm during rainy periods than they did during dry periods.⁸ Seminal plasma from those animals showed higher concentrations of lysine-specific demethylase 5D (KDM5D), a protein that is a key determinant of spermatogenesis,¹⁸ during the rainy period than it did during the dry period.⁸ However, this protein was not identified in the proteome of seminal plasma from peccaries.¹⁹

The characteristics of the fresh sperm collected from collared peccaries were not affected by semiarid weather parameters. This finding contrasts with how climate-related environmental parameters have been observed to affect the reproductive characteristics of peccaries living in tropical²⁰ and semihumid equatorial²¹ climates. In those climates, changes in rainfall index occurring at the peaks of summer and winter correlate with significant changes in ambient temperature that influence the reproductive characteristics of peccaries, including the quality of their sperm. Under a semiarid climate, however, the temperature remains constant across dry and rainy periods and differences in rainfall index and humidity seem to be insufficient to affect the quality of peccaries' fresh sperm. The absence of such an effect reflects that collared peccaries are robust and well-adapted to a semiarid climate of the Caatinga such that the species maintains good reproductive performance throughout the entire year.²²

Interestingly, sperm collected from collared peccaries during the rainy period exhibited better freezability than did sperm collected during the dry period, as reflected by the higher membrane integrity, motility, and rapid subpopulation. Many authors have pointed out that sperm motility is commonly measured to gauge “sperm quality” because it indirectly indicates metabolic activity and sperm membrane integrity.^23,24 In this sense, Campos et al.²⁵ recently demonstrated that sperm motility (r = 63.6%; p < 0.0) and membrane integrity (r = 65.1%; p = 0.03) are highly related to the ability of a peccary's sperm to bind to the zona pellucida of heterologous substrates.

The present findings suggest that a protective factor might exist that is conferred to sperm collected from collared peccaries during the rainy period. In sheep, it is hypothesized that the composition of the seminal plasma may exert a protective effect on sperm during cryopreservation via the presence of specific proteins.^26,27 In domestic swine, the presence of proteins with molecular masses of 18, 19, 44, 65, 80, and 100 kDa in seminal plasma was found to be a potential marker for the tolerance of their sperm for cryopreservation.²⁸

Echoing the findings reported here for sperm from collared peccaries, Aguiar et al.⁷ recently demonstrated that the freezability of sperm from goats bred under a semiarid climate is negatively affected by the dry period. According to the authors, the biochemical components of the goats' seminal plasmas varied across climatic periods, suggesting that the presence of a greater concentration of the enzyme phospholipase A2 in their seminal fluids during the dry period compared with the rainy period might be responsible for reducing the viability of their sperm. A study of peccaries' proteome revealed that phospholipase A2 was not identified,¹⁹ but proteins involved in spermatic protection, such as clusterin and T-complex protein 1, and proteins with antioxidant activities, such as glutathione peroxidase (GPx-5), ceruloplasmin, albumin, and transferrin, were identified in their seminal plasmas. Whether the concentrations or activities of these or other proteins vary across different climatic periods and whether such variation could influence the efficiency of cryopreservation of sperm from peccaries is not known.

In conclusion, the environmental parameters measured in this study did not affect the quality of fresh sperm collected from collared peccaries, but did influence the freezability of sperm collected from collared peccaries raised in a semiarid climate. Therefore, the results of this study suggest that collecting semen from collared peccaries living in a semiarid climate during its rainy periods will benefit efforts to biobank their sperm.

Footnotes

Acknowledgments

The authors thank the Brazilian Council of Scientific Development (CNPq) (Process no.: 407.302/2013-1) for financial support and CEMAS/UFERSA for providing the animals used for this study. A.R.S. received a grant from the CNPq. Others involved received a grant from CAPES.

Author Disclosure Statement

No conflicting financial interests exist.

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