Efeito de dietas contendo caroço de algodão sobre a qualidade do sêmen de ovinos

This study evaluated the semen quality of Santa Ines sheep fed diets containing different levels of whole cottonseed (WCS). Twenty sheep were distributed into four groups that were given diets with 0; 20; 30; 40% of WCS. For this study the semen was collected by electroejaculation, and a monthly andrological examination was performed during nine months. The analyzed variables were: scrotal circumference (SC), appearance and color of the ejaculate, progressive motility (PM), vigor, mass movement (MM), total sperm concentration (spzX109), sperm morphology (SM) and blood hematocrit. The SC had no difference among diets, with mean 31.83 cm. The animals fed diets with WCS, presented mean intake of 9.16 mg gossypol kg BW, and were prone to present greenish semen, and appearance ranging from thin creamy to watery. The PM decreased linearly (p < 0.05) with increasing level of WCS, while the vigor had a quadratic trend. The total defects increased linearly in relation to reference values described in literature. The WSC had no influence on hematocrit levels, but did affect the PM and vigor of spermatozoa, thus not recommended to diets of animals for breeding.


Introduction
The use of cotton derivatives in animal feeding is widespread for the good quality of its protein and other components, such as calcium, phosphorus, iron, and vitamins, besides the energy value.However one of the main problems when using the whole cottonseed or some of its derivatives is the presence of gossypol, an antinutritional factor.The free form of gossypol is predominant in the whole cottonseed (CALHOUN et al., 1995) but during processing to obtain the meal, the gossypol binds to proteins.In the bound form, it is considered less toxic, because its absorption by the digestive tract is impaired (MENA et al., 2001), although there are evidences that the bound form can become free during the fermentation in the rumen (BLACKWELDER et al., 1998).Kerr (1989) observed that all gossypol in the whole cottonseed is found in free form and is readily absorbed after intake and acts on the metabolism of amino acids, binding to proteins with free amino acids.It is important to know the gossypol levels in the diets as increases the amount of dry matter ingested by animals, due to increased production.Randel (1992) mentions that the decrease in sperm motility and sperm count are among negative effects of gossypol.Also Chase Jr. et al. (1994) verified that these reproductive problems are due to degeneration of testicular tissue, resulting in decreased the number of sperm that reach maturity and increased percentage of morphological defects.
The fertility and productivity of a herd are directly associated with the male's reproductive capacity.Thus, the libido, the sperm production and its fertilization power are attributes that define the reproductive efficiency of the animal, which is also subjected to environmental factors, such as temperature, air relative humidity, age, nutrition, and management system (SALGUEIRO; NUNES, 1999).
Given the above, this study evaluated the influence of whole cottonseed on the macroscopic, microscopic and morphology of semen and blood hematocrit of Santa Ines sheep, with breeding age.

Material and methods
The study was conducted at the Experimental Station Pendência, of the State Enterprise for Agricultural Research of Paraíba (EMEPA), in the microregion of 'Cariri paraibano', in the municipality of Soledade, Paraíba State.The trial as performed between October 2004 and June 2005, with average temperature of 30ºC and relative humidity of 60%.
Twenty sheep of Santa Ines breed, aged five months, and initial mean weight of 30.0 ± 2.6 kg.The animals were vaccinated against clostridiosis, wormed and kept in intensive management system in collective pens, with five animals per pen.After the adaptation to installations and management (10 days), the animals were submitted to clinical and andrological examination, aiming to standardize the experimental groups.
Diets have been formulated with increasing levels of whole cottonseed (0, 10, 20 and 30%) in the dry matter, and calculated to meet the protein and energy requirements for gains of 150 g day -1 (NRC, 1985).The compositions of the diets are listed in Table 1.The mean intake of dry matter was determined by the daily control of supplied food and leftovers.The diet was given at 8 am and 4 pm, and the leftovers were weighed and registered on the following morning.Samples of the diets and of the leftovers were gathered fortnightly to analyze: dry matter (DM), crude protein (CP), mineral matter (MM), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), according to Silva and Queiroz (2002).The total gossypol of the cottonseed was analyzed through high pressure liquid chromatography (HPLC) according to described technique (ABOU-DONIA et al., 1981).
In order to estimate the gossypol intake, the WCS was weighed daily and mixed with other ingredients when the diet was supplied, in the subsequent day, we quantified the WCS in the leftovers, determining thus its consumption.
Each month the animals were submitted to clinical and andrological assessment, when the semen was collected through electroejaculation, using an automatic electroejaculator (Autojact, Neovet) with 12 V and 5 A, the ejaculates were obtained by the automatic method with animals standing in a sheep chute, followed by a sequence of 1-35 stimuli, adding up a total of 30 seconds to 5 minutes.The volume of the ejaculate was measured by means of a collection tube, which was usually 1.2 mL per ejaculate.After this, it was kept in a water bath at 37ºC, and we evaluated the macroscopic (volume, appearance and color) and microscopic characteristics (mass movement, progressive motility, vigor, sperm morphology and concentration).Blood samples were obtained by puncturing the external jugular vein in heparinized tubes at the beginning, middle and end of the trial, in order to determine the hematocrit levels in the animals' blood.The measures of the testis were made during semen collection, the scrotal circumference was assessed using a measuring tape surrounding the central surface of the testicles and expressed in centimeters.The length of the testis was made using a caliper open between the caudal end of the head and the cranial end of the tail of the epididymis and expressed in centimeters.
Other assessments were made in the testis, like symmetry (symmetrical standard), shape (ovoid standard), position (vertical standard), consistence (turgid elastic standard), sensitivity (absent standard) and motility (present standard).The epididymis was evaluated by palpation of the presence and integrity of its parts, head, body and tail; and the scrotum, by visualization of lack of continuity solutions or diverse injuries.
Considering the semen, it was evaluated its volume soon after obtaining the sample, through direct reading of millimeter graduation of the collection vial, and the result was expressed in milliliter (mL).The sample was then kept in water bath at 37°C, and the evaluations were made in sequence, according to CBRA (1998).
The appearance of the semen was determined by visualization of consistence of ejaculates and classified as: creamy marble, creamy, thick milky, milky and watered.To evaluate the mass movement, a 10 μL-aliquot was placed on preheated slides (37ºC) and observed under optical microscope (100 x magnification).The result was expressed in an increasing scale of 0-5 points.
The progressive motility and vigor were determined by placing 10 μL of semen into 1 mL of Tris dilution buffer (Hydroxymethylaminomethane) (3.0 g), sodium citrate (2.0 g) and fructose (1.0 g).Then a 10 μL-aliquot of the diluted semen sample was placed between preheated slide and coverslip (37ºC) and evaluated under optical microscope (100 x magnification).The progressive motility was expressed in percentage and the vigor in an increasing scale of 0-5 points.
Sperm concentration was determined by direct counting of spermatozoids in a Neubauer chamber, by diluting a sample of 20 μL into 2 mL of distilled water, obtaining a final dilution of 1:200, being the result presented in mm 3 .
Sperm morphology was performed from 95 smears stained with Giemsa at 7.5% (Doles Laboratory), diluted in distilled water and immersing the slides in this solution for two hours.Afterwards the slides were kept upright until completely dry.It was counted 100 sperm cells of each smear, and the result was expressed in percentage.
A completely randomized design was used, with four diets and five replicates.The data of microscopic characteristics of the semen was submitted to an analysis of variance and regression analysis, as a function of the whole cottonseed levels.Statistical analysis were made using the software SAS (Statistical Analysis System) described by (SAS, 1999).The subjective variables, color and appearance, are presented only for description, they were not included in the statistical models.

Results and discussion
Table 2 presents the data about the performance of the animals and their mean intake, in kg day -1 of dry matter, of whole cottonseed and gossypol in mg kg -1 LW.The mean concentration of gossypol in the whole cottonseed was 1,547 mg kg -1 in the dry matter.Considering that the sheep had a mean intake of 0.249, 0.320 and 0.399 kg day -1 of whole cottonseed, the estimated intake of gossypol corresponded to 6.8, 9.2 or 11.5 mg kg -1 LW in diets with 20, 30 or 40% of whole cottonseed.
The daily weight gain, expressed in g day -1 , had no influence from the levels of whole cottonseed, with a mean value of 164.0 g day -1 , indicating that the animals presented a good development of weight throughout the study period.The mean daily intake of dry matter was similar for the four levels of whole cottonseed, with mean values of 1.919 kg and 3.72% of live weight, respectively.For young ruminants, Tool Vera (1997) mentions that the maximum amount of gossypol should be 10-20 mg kg -1 .In this study, the animals that had consumed 9.20 or 11.50 mg gossypol kg -1 LW day -1 , presented the motility and vigor affected (Table 3) in relation to the control diet.
Scrotal circumference (SC) has high correlation with sperm production, service capacity and sexual development (YARNEY et al., 1990).In this study, the SC and the length of the testis were not influenced by the levels of whole cottonseed, whose mean values were 31.84 and 1.92 cm, respectively (Table 3) and the animals presented symmetrical testes, with turgid elastic consistence, no anomalies and mobile within the scrotum.
These results diverge from those found by Andreazzi et al. (1995), working with animals since weaning with 20-30 days until 18 months of age, which observed smaller scrotal circumference in goats receiving 30% of whole cottonseed in relation to the control diet.The results with sheep may have been influenced by age, once the animals were in process of growth during the experimental period.Therefore, mean values of 34 and 36 cm are expected for SC of adult sheep (NUNES et al., 1998;BITTENCOURT et al., 2004).
Similar results were obtained by Kramer et al. (1991), with pubescent rams receiving gossypol in the diet.In the same way, Jimenez et al. ( 1989), working with bulls, observed that gossypol intake had no influence on the SC, compared to the control animals.
Macroscopic characteristics of the semen, like color and appearance, are described just for information.On average, the semen had consistency from thin creamy to watery.As for the color, the animals that ingested higher levels of whole cottonseed had presented greenish semen, from the fourth month of supply the diets, pointing out the effect of gossypol on these characteristics.Under normal conditions, the semen of sheep is creamy and white.
Microscopic characteristics (Table 3) referring to volume of ejaculate, mass movement, and sperm concentration, were not influenced by the diet, whose mean values were 1.45 mL; 2.65 (0-5) and 2.16 (10 9 spz mL -1 ), respectively.The values for the volume were higher than reported by Tutida et al. (1999), in sheep of different genetic groups, and similar to those found by Salgueiro and Nunes (1999), in Santa Ines sheep, being within the range observed in literature for the species, 0.5 to 1.5 mL (CBRA, 1998).Furthermore, Ferreira et al. (1995) obtained values of 0.89, 0.86 and 1.03 mL for the levels of 0, 30 and 60% of cottonseed meal in the diet of goats.However, as this is a subjective assessment, the values found in literature are very variable and can only be observed for species with highly concentrated semen.
In relation to sperm concentration, Boundy (1993) affirmed that a good quality semen must contain 3.5-6.0 million sperm per milliliter of ejaculate.Our results were below these values, but were similar to those reported by Moreira et al. (2001) for Santa Ines sheep.
Freitas and Nunes (1992) studying sheep in the Northeastern region, observed values of 3.98 x 10 6 mm -3 during dry season, and 3.41 x 10 6 mm -3 during rainy season.
The progressive motility and vigor of sperm cells had been influenced by the whole cottonseed (p < 0.01), with a downward linear trend as increased the WCS level.The results corroborated the results of Risco et al. (1993) that fed bulls with a diet without gossypol and another diet with 8.2 g day -1 of free gossypol, and verified that the sperm motility was affected (52.0 ± 9.8 vs. 82.0± 6.2%) in relation to the control group, in the ninth week, whereas the percentage of normal sperm decreased from the fifth week (49.0 ± 9.8 vs. 82.0± 3.2%), and still the authors observed erythrocyte fragility.Moreover, Santos et al. (2008) registered a reduction in sperm motility (from 73.7 to 52.5%) and an increase of total defects of sperm cells (26.8 x 20.0%), respectively for bulls fed diet with no gossypol and diet with 2.2 kg of whole cottonseed.The sperm vigor presented quadratic trend, whose maximum was 3.3 points for the control diet.This indicates that this characteristic was affected by the WCS, since these values were below the average of 3 points, cited by CBRA (1998) as a desirable standard to select animals for breeding.Maugh (1981) evidenced that the gossypol alters the spermatogenesis, causing oligospermia and aspermia, by inhibiting the lactate dehydrogenase X, an enzyme that plays a key role in spermatozoa metabolism.Chase Jr. et al. (1994) fed growing cattle diets with 0, 6 or 60 mg kg -1 LW, for 196 days and verified delayed puberty in the animals treated with whole cottonseed, in relation to the control group; however the quality and quantity of semen was not different between the treatments.Nevertheless, the authors mentioned an increase in the lumen diameter, with lower density of germinal epithelium, and lower amount of Leydig cells in the animals that received gossypol, leading to the conclusion that the gossypol did not affect the quality of the semen, but caused damages to testicular morphology.
The results permitted to infer a probable effect of gossypol, since the literature cites that its concentration in the plant has negative correlation with environmental temperature, and positive with rainfall (KERR, 1989).However, in this study it was not possible to determine the action of gossypol for affected variables; since testosterone and plasma gossypol have not been dosed.Zahid et al. (2003) fed goats with gossypol and observed that it did not affect the semen quality, but rather had affected significantly (p < 0.05) the pH, motility, and percentage of morphologically abnormal spermatozoa.
By observing the sperm morphology (Table 4), the percentage of total defects had increased linearly.The regression analysis allowed verifying that, for each 1% increase of whole cottonseed in the diet, there was an increase of 0.2% in total defects.Meantime, these values were below the limit of 20% for sperm abnormality cited by (CBRA, 1998).The morphological analyses using Giemsa staining stood out a greater occurrence of major defects, such as swelling of the acrosome, folded tail, and strongly folded tail.In accordance with Bittencourt et al. (2004), one of the most important factors when evaluating the morphology is the acrosome integrity, which is indispensable for a high fertilization.The results were lower than found by Santos et al. 2008) that achieved a mean value of 12.2% for major defects, and 20.2% for total defects, in Nelore bulls fed diets with whole cottonseed.Chenoweth et al. (1994) working with Young cattle, a group without gossypol, and another receiving 8.2% of free gossypol a day, for 11 weeks, have concluded that the supplementation with cottonseed meal had adverse effect on the sperm morphology and spermatogenesis, with the first alterations detected between the 3 rd and 4 th weeks of the established diet.Additionally, Arshami and Ruttle, (1989), feeding sheep with 0 or 12% of cottonseed meal in the diet, for 26 weeks, have verified abnormalities, such as the wider diameter of the lumen, low number of Leydig cells, smaller size of Sertoli cells, and thinner walls of seminiferous tubules.Through the analysis of the Table 5, we can infer that the whole cottonseed had no influence on the hematocrit levels, which remained within the reference values for sheep (24-50%).However it was observed a drop in the values in the final phase, corresponding to the last assessment.The mechanism whereby the gossypol interfere on erythrocyte fragility is not yet fully understood, but it is speculated that it may interact directly with the membrane of red blood cells (HARVEY, 1989), and changes their fluidity (VELÁSQUEZ-PEREIRA et al., 1999).Beaudoin (1985), analyzing ruminants receiving diets with cottonseed meal or diets with whole cottonseed cited a reduction in the blood hemoglobin rate.On the other hand, Risco et al. (1993) stated that the changes observed in hematological and serum composition of cows fed with 0 or 800 mg of free gossypol (FG kg -1 ) have not been enough to be used as a diagnosis of toxicity.Likewise, Andreazzi et al. (1995) did not detect significant difference between the treatments for the blood metabolites in goats fed with 0 or 13.45 mg of FG kg -1 LW.Besides that, Calhoun et al. (1995) proposed that the maximum safe concentration of gossypol in the plasma should be 5 μg mL -1 .
In ruminants, Mena et al. (2001) argued that the detoxification in the rumen is reduced when increased the ruminal passage rate of foods with gossypol.
However Guedes and Soto-Blanco (2010) said gossypol is a compound highly reactive that binds rapidly to different substances, including minerals and amino acids.In males, gossypol promotes reduction of motility and spermatozoid concentration.

Conclusion
The use of whole cottonseed in diets for sheep influences the quality of the semen, especially the progressive motility and vigor, leading to an increase in the percentage of total defects in the sheep semen; therefore it should not be supplied to animals for breeding.

Table 2 .
Mean value, regression equation, coefficient of variation (CV) of the performance and intake of food of Santa Ines sheep fed whole cottonseed.

Table 3 .
Mean value, regression equation, coefficient of variation (CV) and of determination of reproductive parameters of Santa Ines sheep fed whole cottonseed.

Table 4 .
Sperm morphology in Santa Ines sheeps fed diets with whole cottonseed.

Table 5 .
Mean values of blood hematocrit in Santa Ines sheep fed diets containing whole cottonseed.