Sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the Brazilian Northeast: the season's influence on the cooling of semen

Caracrterísticas espermáticas e bioquímicas do plasma seminal de caprinos nas estações chuvosa e seca do Nordeste brasileiro: influência da estação no resfriamento do sêmen

VETERINARY MEDICINE

Sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the Brazilian Northeast: the season's influence on the cooling of semen

Caracrterísticas espermáticas e bioquímicas do plasma seminal de caprinos nas estações chuvosa e seca do Nordeste brasileiro: influência da estação no resfriamento do sêmen

G.V. Aguiar^I; M.F. van Tilburg^I; A.G.V. Catunda^I; C.K.S. Celes^I; I.C.S. Lima^I; A.C.N. Campos^I; A.A.A. Moura^I,*; A.A. Araújo^II

^IUniversidade Federal do Ceará - Fortaleza, CE

^IIUniversidade Estadual do Ceará - Fortaleza, CE

ABSTRACT

The present study aimed to verify the caprine semen characteristics during dry and rainy seasons in the Brazilian Northeast, and the influence of these seasons on cooled semen. Seminal volume, concentration, percentage of motile cells, vigor and spermatic morphology, as well as biochemical profile (fructose, citric acid, P, Ca²⁺, Mg, total proteins and phospholipase A₂ activity) were analyzed. It was observed a reduction (P<0.05) in normal sperm morphology, fructose, citric acid, P, Mg and total protein concentration during the dry season, which did not affect the motility, vigor, volume and sperm concentration. Phospholipase A₂ activity was increased during the dry season (P<0.05). The analysis of the semen cooled at 4ºC during 48 hours showed reduction in total motility and vigor sperm during the dry season (P<0.05). Based on these results, we conclude that the best period of year for caprine semen cooling is the rainy season.

Keywords: semen, spermatozoa, season, cooling, caprine

RESUMO

Verificou-se as características seminais de caprinos durante a época seca e a chuvosa no Nordeste brasileiro e a influência da época no resfriamento do sêmen. Foram mensurados volume, concentração espermática, porcentagem de espermatozoides móveis, vigor, morfologia espermática e características bioquímicas (frutose, ácido cítrico, fósforo, magnésio, proteínas totais e atividade da fosfolipase A₂). Observou-se redução (P<0,05) no número de espermatozóides morfologicamente normais, frutose, ácido cítrico, fósforo, magnésio e proteínas totais durante a época seca que não influenciaram na motilidade, vigor, volume e concentração do sêmen. Entretanto, a atividade da fosfolipase A₂ foi maior na época seca. Quando o sêmen foi submetido ao resfriamento a 4ºC durante 48 horas, houve redução (P<0,05) na motilidade total e no vigor espermático durante a época seca. Com base nesses resultados, conclui-se que o período chuvoso é melhor para resfriar sêmen de caprinos no Nordeste brasileiro.

Palavras-chave: sêmen, espermatozoide, época do ano, resfriamento, caprino

INTRODUCTION

In the last decades, research has focused on the improvement of biotechnology for animal breeding and methods of semen preservation for artificial insemination, embryo transfer and in vitro fertilization (Trummer et al., 1998; Holt, 2000; Yoshida, 2000; Pauw et al., 2003). In this regard, there are also specific approaches for defining the best method, interval and time of year for collection and preservation of semen samples (Amir et al., 1986; Shamsuddin et al., 2000; Campos et al., 2003), as well as appropriate strategies for the dilution of those samples (Watson, 2000) and effective use of extenders (Singh et al. 1995; Gil et al., 2003; Eiman and Terada, 2004).

The reproductive performance of goat herds directly depends on the genetic potential of livestock, management and environment. The interrelationship of these factors determines the adaptation of the animals and their reproductive efficiency (Robertshaw, 1982). In temperate regions, animals with seasonal reproductive characteristics are influenced by a combination of photoperiod and temperature, while in tropical regions the environmental effect seems to be related to rain and its effect on the amount and quality of forage (Rege et al., 2000). However, other climate factors such as humidity and temperature changes can cause thermal discomfort, resulting in a decrease in food intake and interference with spermatogenesis and semen quality (Kunavongkrit et al., 2005).

Thus, this study was designed to evaluate the effect of seasons, defined as rainy and dry, in the cooling of caprine semen, sperm parameters (vigor, percentage of motile cells, degradation rate of sperm motility and morphology) and seminal plasma composition (calcium, phosphorus, magnesium, fructose, citric acid and total protein, PLA2 activity) in goats raised in the Northeast of Brazil.

MATERIALS AND METHODS

The experiment was conducted in the Northeast of Brazil, at 3º45' South and 38º32' West, 15.5m above sea level. The weather in this region is defined by Koppen as hot and humid (AW), with average temperature between 26 and 27ºC, maximum of 30ºC and minimum of 19ºC. The period of the study was year 2006, including a rainy season from March to May and a dry season from September to November. The former was characterized by an average rain fall of 345±123.4mm/year, humidity of 82.3±3.2% and temperature of 27±3.2ºC, varying from 24.4±0.4 to 30.3±0.4. In contrast, the dry season had an average rain fall of 5.4±4.3 mm/year, humidity of 69±1% and temperature of 27.6±0.1ºC, with amplitude from 25.2±0.3 to 31.1±0.2. The temperature and humidity index (THI) was determined as 75.1±1.2 for the rainy season, with a range from 73.6 to 76.8, and 75.4±1.5 for the dry season of the year, ranging from 73.3 to 77.5 (Silanikove, 2000).

We used 17 non-defined breed goats, with 26.2±5.4 months of age and 41.1±4.2kg. During the experiment, animals were raised in individual pens and fed Tifton hay (Cynodon dactilum) and concentrate (250g/head/day), following NRC (National..., 1981) recommendations. Semen was collected weekly for seminal plasma and biweekly for analysis of sperm parameters, using an artificial vagina model designed for sheep (Walmur^®, Porto Alegre, RS, Brazil). In the first case, semen was centrifuged at 700g for 20 minutes (4ºC) and the supernant seminal plasma stored at -18ºC until use for biochemical analysis, as reported previously (Souza et al., 2010). In the case of samples collected biweekly, we recorded the ejaculated volume and diluted a 50-µL aliquot of semen in a phormol-saline solution (0.1% v/v) to determine sperm concentration using a spectrophotometer (Spectrophotometer SP M05 Bel^® Photonics), according to Chemineau et al. (1991). The sperm concentration was measured and the semen samples were diluted in coconut water solution (ACP^®) to obtain a final concentration of 2x10⁸ sperm/mL. From this solution, an aliquot was used to estimate the percentage of motile sperm and vigor, using a scale from 0 to 5 (Chemineau et al., 1991).

The media used to dilute semen samples was initially prepared as a stock solution, containing 50% of coconut water solution (ACP^®) and 50% citrate (2.5%), pH 6.2-6.8 and osmolarity at 300mOsmol/L. Immediately before mixing with semen samples, 2.5% of egg yolk was added to this stock solution (Cardozo et al, 2006). After the final dilution, semen samples were incubated at 4ºC and frozen for 48 hours. During this period, sperm vigor and percentage of motile cells was determined at 2, 24 and 48 hours. Additionally, we conducted the thermoresistance test (TRT), which consisted of incubating 250µL of semen at 38ºC, during 5 and 120 minutes, according to Chemineau et al. (1991). Semen aliquots were then collected, stained with bromophenol blue (1g bromophenol blue, 4g sodium citrate to 100mL distilled water) in glass slides and sperm morphology analyzed (200 cells/ejaculate; Derivaux, 1980). The degradation motility rate (DMR) was calculated at the end of the TRT, according to the formula: DMR = (sperm vigor at 5min. – sperm vigor at 120min.)/(sperm vigor at 5min.) x 100.

Considering the low volume of seminal plasma obtained from each ejaculation, all samples collected in each month were pooled. Thus, this approach allowed us to have enough volume for all analysis of the seminal plasma components. Concentrations of Ca²⁺, P, Mg and total protein were determined using commercial kits (Labtest Diagnóstica SA, Lagoa Santa, MG, Brazil) and those of citric acid and fructose, using Espermoteste® kits (InVitro Diagnostic S/A, Itabira-MG, Brazil), as described previously (Catunda et al., 2009). Phospholipase A₂ activity was quantified according to adaptations of the method published by De Haas et al. (1968). First, we prepared a solution containing one egg yolk in 50mL of distilled water. From this stock, 15mL was removed, mixed with 10mL of 0.03M sodium deoxycholate, 1mL of 0.6M CaCl₂ and distilled water q.s.q. in 100mL, pH 7.8-8.0. A 10mL aliquot of this final solution was added to 20µL of goat seminal plasma and pH was measured (PHS-3B, pHTEK). We analyzed the time required for a 0.03 pH unit decrease and then added 3µL of 0.11N NaOH. The methodology was carried out continuously during three minutes. After this, we averaged the time gaps between each time measured during the assay. This average was in turn multiplied by a correction factor to determine the total consumption of NaOH/minute/each 20µL aliquot of seminal plasma. For every sample, phospholipase A₂ activity was determined three times and the average of these repetitions was used for statistical analysis.

Variations in semen parameters and components of seminal plasma as related to season of the year were evaluated by analysis of variance and Tukey statistical test SAS (Statistical..., 2008). Variables defined as sperm motility and DMR were transformed in arcsen to fit normality requirements. Also, we used Pearson's method to estimate the correlations among sperm parameters and seminal plasma components (fructose, citric acid, total protein, Ca^2+, P, Mg, and PLA₂ activity) within each season of the year SAS (Statistical..., 2008). Correlations were considered significant with p values <0.05.

RESULTS

In the case of fresh semen samples, the percentage of sperm with normal morphology was significantly higher (P<0.001) during the rainy season when compared to those collected in the dry season, but ejaculate volume, sperm motility, vigor and sperm concentrations did not differ between those periods of the year (P<0.05; Table 1).

When semen samples were subjected to cooling for 2, 24 and 48 hours, the percentage of motile cells and sperm vigor were always lower in the dry as compared to the wet season (P<0.05; Table 2). Moreover, the degradation motility rate (DMR) measured at 2, 24 and 48 hours of cooling time was 2, 1.8 and 1.6-fold higher in the dry than in the rainy season (P<0.05). Sperm morphology, determined in semen samples subjected to cooling, was different between dry and rainy seasons but not associated with cooling time regardless of the period of the year (Table 2).

Concentrations of Ca²⁺ remained without major changes throughout the study (P>0.05) and those of fructose, citric acid, P, Mg and total protein in the goat seminal plasma were consistently lower in the dry season compared to the rainy season (P<0.05). The greatest variations were associated with the concentrations of P and fructose, representing 26.8 and 31.3 % less in the dry when contrasted with the rainy season, respectively. Phospholipase A₂ activity was 32 % higher in the dry season when compared to the rainy period (P<0.05; Table 3).

DISCUSSION

The present study describes the variations of goat semen criteria and certain components of seminal plasma associated with periods of the year characterized by pronounced differences in rain fall. The region where those animals were raised, the Northeast of Brazil, was at 3^o45' of latitude, thus with no significant changes in day length and where seasons were defined mainly as dry and rainy. The former usually occurs from March to May, equivalent to autumn, and the later takes place from September through November, timely with the spring season in the Southern hemisphere.

Parameters such as volume of ejaculates, sperm concentration, percentage of motile cells and sperm vigor in fresh semen samples did not show significant differences related to periods of the year, but the number of sperm with normal morphology decreased in the dry season. However, when semen samples were subjected to cooling up to 48 hours, more specific and significant differences became evident between dry and wet seasons. In this case, the percentage of motile cells, sperm vigor and the number of morphologically normal cells were lower while DMR was higher in the dry season as compared to the wet period of the year.

The fact that sperm parameters in fresh semen did not differ between seasons cannot be associated with nutritional factors because all animals were confined and received the same diet during the experiment. However, there was a small increase in sperm pathology during the dry season, which can be due to the higher air temperatures reached during that period. Results similar to the ones presented here were found by Dias et al. (1995) and Vieira et al. (2008), who also reported significant increases in sperm pathology in semen of goats during the drought seasons in the Midwestern and Northeastern Brazil, respectively. As well established, heat stress triggers alterations in the seminiferous epithelium, also with deleterious effects on semen quality. Experimentally induced elevation of testicular temperature causes significant increases in sperm pathology (Lagerlof, 1938) and, with the rising temperature of the normal testis, as observed in cryptorchid animals (Moretti et al., 2007) or experimental-induced heat stressed testes (Moreira et al., 2001), spermatogenesis can be completely arrested. The severity of testicular degeneration depends on time and temperature of exposure, but even an increase of 1 or 2ºC for eight hours can cause marked changes in the spermatogenesis (Entwistle, 1992).

In the present study, when semen samples were subjected to cooling at 4ºC, there was a significant decrease in the percentage of motile cells and vigor throughout the cooling time in both seasons. The cooling process can cause irreversible damage to sperm due to heat shock, which can be minimized by the slow cooling of diluted semen in a ratio of 0.05ºC/min to 4ºC (Pickett and Amann, 1993) and by adding extenders containing phospholipids, such as milk or egg yolk (Amann and Pickett, 1987; Den Daas, 1992). However, the dilution of goat semen in extenders containing egg yolk can be deleterious to sperm cells. This occurs because the goat semen presents characteristics that differentiate it from other species, being the most important the presence of phospholipase A, secreted by the bulbourethral glands. This phopholipase is also called Eyce (Egg yolk coagulating enzyme) or BUSgp60 (bulb urethral gland secretion; LeBoeuf et al., 2000) and is responsible for the reduced viability of sperm cells that have been cooled or frozen in extenders containing egg yolk or milk, respectively (Corteell, 1981).

Considering the absence of significant differences related to sperm motility and vigor of semen collected before cooling, we also suggest that differences associated with the cooling time between the dry and rainy seasons relate to biochemical changes found in seminal plasma in the respective periods. In fact, we observed significant reductions in phosphorus, magnesium, citric acid, fructose and total proteins in the seminal plasma collected in the dry when compared to the wet season. Changes in biochemical components of the goat semen as related to seasons of the year were also observed by Roca et al. (1993) and Catunda et al. (2009) and it is well known that minerals are important for electrolytic balance and essential for the conservation of goat semen. These components also play a diverse range of roles on reproductive systems, such as regulation of intra and extracellular enzymes, membrane proteins, second messengers, receptors and energy metabolism (Smith and Akinbamijo, 2000).

Most extenders use the composition of egg yolk as a basic component, since the protection of spermatozoa against thermal shock is favored by phosphatidylcholines (lecithins), the lipoprotein yolk and milk casein (Dee Haas, 1992). However, the dilution of goat semen in extenders containing egg yolk can be deleterious to sperm cells. This fact can be observed when comparing the degradation motility rate (DMR) with two hours of cooling in the dry and rainy seasons. The significant increase of DMR in the dry season is probably related to increased phospholipase A2 activity present in seminal plasma, which induced a reduction in sperm motility and vigor in the same period. The negative influence exerted by the phospholipases present in seminal plasma persists even at temperatures of cooling or freezing. Thus, according Corteel (1974), there was a reduction in the number of live sperm directly proportional to storage time. The presence of phospholipase in goat seminal plasma, as previously mentioned, is unfavorable to its conservation, either when cooled or frozen.

CONCLUSION

Results obtained in the present study indicate that native goats of non-defined breed, raised in a tropical region of Brazil, show variations in the biochemical components of seminal plasma during the dry and rainy seasons. In fresh semen, sperm morphology was the only parameter meaningfully affected by season. However, when semen was subjected to cooling with extender containing egg yolk, sperm motility was negatively affected in the dry season of the year. This fact was coincident with higher phospholipase A₂ activity present in seminal plasma during the dry season, suggesting that this enzyme triggered the reduction in sperm viability.

Recebido em 14 de outubro de 2011

Aceito em 28 de agosto de 2012

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Publication Dates

• Publication in this collection
  05 Mar 2013
• Date of issue
  Feb 2013

History

• Received
  14 Oct 2011
• Accepted
  28 Aug 2012